Methods and systems for multiplexing of droplet based assays

ABSTRACT

Compositions, methods, and systems are provided for sample preparation techniques and sequencing of macromolecular constituents derived from a cell (i.e., a cell bead) in a multiplexed reaction. Using the compositions, systems, and methods disclosed herein, the association of the macromolecular constituents with the biological particle from which they are derived and the association of the cell bead with the cell bead sample from which they are derived is maintained.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/790,358 filed Jan. 9, 2019, which application is entirelyincorporated herein by reference.

BACKGROUND

A sample may be processed for various purposes, such as multiplexingassays to associate a cell type with nucleic acids associated with thatcell type. The sample may be a biological sample. Biological samples maybe processed, such as for detection of a disease (e.g., cancer) oridentification of a particular species. There are various approaches forprocessing samples, such as polymerase chain reaction (PCR) andsequencing.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Biological samples in partitions may be subjected to various processes,such as chemical processes or physical processes. Samples in partitionsmay be subjected to heating or cooling, or chemical reactions, such asto yield species that may be qualitatively or quantitatively processed.

SUMMARY

In an aspect, provided is a method for cellular analysis, comprising (a)providing a first cell bead comprising (i) a first biological particle;and (ii) a first cell bead index molecule comprising a first cell beadindex sequence; (b) providing a second cell bead comprising (i) a secondbiological particle; and (ii) a second cell bead index moleculecomprising a second cell bead index sequence different than the firstcell bead index sequence; (c) pooling the first cell bead and the secondcell bead to provide a pooled plurality of cell beads; and (d)partitioning the pooled plurality of cell beads into a plurality ofpartitions to generate (i) a first partition comprising the first cellbead and a nucleic acid molecule comprising a first barcode sequence and(ii) a second partition comprising the second cell bead and a nucleicacid molecule comprising a second barcode sequence, wherein the secondbarcode sequence is different than the first barcode sequence.

In some embodiments, the method further comprises, (e) generating (i) afirst nucleic acid molecule comprising the first cell bead indexsequence and the first barcode sequence and (ii) a second nucleic acidmolecule comprising the second cell bead index sequence and the secondbarcode sequence.

In some embodiments, the first cell bead index sequence identifies thefirst cell bead and wherein the second cell bead index sequenceidentifies the second cell bead.

In some embodiments, (a) comprises generating a first plurality of cellbeads, including the first cell bead, and wherein each cell bead of thefirst plurality of cell beads comprises a biological particle and thefirst cell bead index sequence. In some embodiments, (b) comprisesgenerating a second plurality of cell beads, including the second cellbead, wherein each cell bead of the second plurality of cell beadscomprises a biological particle and the second cell bead index sequence.In some embodiments, (c) comprises pooling the first plurality of cellbeads and the second plurality of cell beads to provide the pooledplurality of cell beads.

In some embodiments, the method further comprises generating a firstbarcoded molecule comprising a sequence of a cellular nucleic acidmolecule of the first biological particle and the first barcodesequence. In some embodiments, the method further comprises generating asecond barcoded molecule comprising a sequence of a cellular nucleicacid molecule of the second biological particle and the second barcodesequence. In some embodiments, the first barcoded molecule is generatedin the first partition. In some embodiments, the second barcodedmolecule is generated in the second partition.

In some embodiments, the first cell bead and the second cell bead eachcomprises a respective polymer or crosslinked matrix and wherein thefirst cell bead index molecule and/or the second cell bead indexmolecule are embedded within respective polymer or crosslinked matrices.

In some embodiments, the first cell bead and the second cell bead eachcomprises a respective polymer or crosslinked matrix and wherein thefirst cell bead index molecule and/or the second cell bead indexmolecule are covalently attached to respective polymer or crosslinkedmatrices. In some embodiments, the first cell bead index molecule and/orthe second cell bead index molecule are releasably attached to therespective polymer or crosslinked matrices. In some embodiments, thefirst cell bead index molecule and the second cell bead index moleculeare releasably attached to the respective polymer or crosslinkedmatrices by a labile bond selected from the group consisting of athermally labile bond, a chemically liable bond, an enzymatically labilebond, and a photolabile bond.

In some embodiments, the first cell bead index molecule and/or thesecond cell bead index molecule are double-stranded or partiallydouble-stranded.

In some embodiments, the first cell bead index molecule and/or thesecond cell bead index molecule are single-stranded.

In some embodiments, the first cell bead index molecule comprises asequence complementary to a sequence of the nucleic acid moleculecomprising the first barcode sequence and wherein the second cell beadindex molecule comprises a sequence complementary to a sequence of thenucleic acid molecule comprising the second barcode sequence. In someembodiments, the first cell bead index molecule and the second cell beadindex molecule each comprises a poly-A sequence and wherein the nucleicacid molecule comprising the first barcode sequence and the nucleic acidmolecule comprising the second barcode sequence each comprises a poly-Tsequence.

In some embodiments, the method further comprises providing a third cellbead comprising a third biological particle and a third cell bead indexmolecule comprising a third cell bead index sequence different than thefirst and the second cell bead index sequences. In some embodiments, thethird cell bead index sequence identifies the third cell bead. In someembodiments, the method further comprises generating a third pluralityof cell beads, including the third cell bead, wherein each cell bead ofthe third plurality of cell beads comprises a biological particle andthe third cell bead index molecule. In some embodiments, (c) furthercomprises pooling the first plurality of cell beads, the secondplurality of cell beads, and the third plurality of cell beads toprovide the pooled plurality of cell beads. In some embodiments, (d)further comprises generating a third partition comprising a third cellbead of the third plurality of cell beads and a nucleic acid moleculecomprising a third barcode sequence, wherein the first and the secondbarcode sequence is different than the third barcode sequence. In someembodiments, (e) further comprises generating a third nucleic acidmolecule comprising the third cell bead index sequence and the thirdbarcode sequence. In some embodiments, the method further comprisesgenerating a third barcoded molecule comprising a sequence of a cellularnucleic acid molecule of the third biological particle and the thirdbarcode sequence.

In some embodiments, the cellular nucleic acid molecule is adeoxyribonucleic acid (DNA) molecule. In some embodiments, the DNAmolecule is a genomic DNA (gDNA) molecule. In some embodiments, the gDNAmolecule is present in chromatin. In some embodiments, the first, thesecond, or the third barcoded molecule comprises a region of accessiblechromatin.

In some embodiments, the cellular nucleic acid molecule is a ribonucleicacid (RNA) molecule. In some embodiments, the RNA molecule is amessenger RNA (mRNA) molecule.

In some embodiments, the nucleic acid molecule comprising the firstbarcode sequence is attached to a first bead, wherein the nucleic acidmolecule comprising the second barcode sequence is attached to a secondbead, and wherein the first partition comprises the first bead and thesecond partition comprises the second bead. In some embodiments, thenucleic acid molecule comprising the first barcode sequence isreleasably attached to the first bead and wherein the nucleic acidmolecule comprising the second barcode sequence is releasably attachedto the second bead. In some embodiments, the first barcode sequence isreleasably attached to the first bead by a labile bond, wherein thenucleic acid molecule comprising the second barcode sequence isreleasably attached to the second bead by a labile bond, and wherein thelabile bond is selected from the group consisting of a thermally labilebond, a chemically liable bond, an enzymatically labile bond, and aphotolabile bond.

In some embodiments, the first bead and the second bead are each a gelbead. In some embodiments, each of the first bead and the second beadare a degradable gel bead. In some embodiments, each of the first beadand the second bead are degradable upon application of a stimulus. Insome embodiments, the stimulus is a thermal stimulus, a chemicalstimulus, or a photostimulus. In some embodiments, the first partitionand the second partition each comprises the stimulus.

In some embodiments, the first biological particle is a first cell or aderivative of the first cell. In some embodiments, the second biologicalparticle is a second cell or a derivative of the second cell.

In some embodiments, the first biological particle is a first nucleus ora derivative of the first nucleus. In some embodiments, the secondbiological particle is a second nucleus or a derivative of the secondnucleus.

In another aspect, provided is a composition, comprising: (a) a firstcell bead comprising (i) a first biological particle; and (ii) a firstcell bead index molecule comprising a first cell bead index sequence;and (b) a second cell bead comprising (i) a second biological particle;and (ii) a second cell bead index molecule comprising a second cell beadindex sequence different than the first cell bead index sequence.

In some embodiments, the composition further comprises (a) a firstplurality of cell beads, including the first cell bead, wherein eachcell bead of the plurality of cell beads comprises (i) a biologicalparticle; and (ii) a first cell bead index molecule comprising the firstcell bead index sequence; and (b) a second plurality of cell beads,including the second cell bead, wherein each cell bead of the pluralityof cell beads comprises (i) a biological particle; and (ii) a secondcell bead index molecule comprising the second cell bead index sequencedifferent than the first cell bead index sequence.

In some embodiments, the first cell bead and the second cell bead eachcomprises a respective polymer or crosslinked matrix and wherein thefirst cell bead index molecule and/or the second cell bead indexmolecule are embedded within respective polymer or crosslinked matrices.

In some embodiments, the first cell bead and the second cell bead eachcomprises a respective polymer or crosslinked matrix and wherein thefirst cell bead index molecule and/or the second cell bead indexmolecule are covalently attached to respective polymer or crosslinkedmatrices. In some embodiments, the first cell bead index molecule and/orthe second cell bead index molecule are releasably attached to therespective polymer or crosslinked matrices. In some embodiments, thefirst cell bead index molecule and the second cell bead index moleculeare releasably attached to the respective polymer or crosslinkedmatrices by a labile bond selected from the group consisting of athermally labile bond, a chemically liable bond, an enzymatically labilebond, and a photolabile bond.

In some embodiments, the first cell bead index molecule and/or thesecond cell bead index molecule are double-stranded.

In some embodiments, the first cell bead index molecule and/or thesecond cell bead index molecule are single-stranded.

In some embodiments, the first cell bead index molecule and the secondcell bead index molecule each comprises a poly-A sequence.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles or cell beads.

FIG. 2 shows an example of a microfluidic channel structure forco-partitioning cell beads and barcode beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning biological particles or cell beads and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

FIG. 8 illustrates an example of a barcode carrying bead or cell beadwith cell bead index molecules.

FIG. 9 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

FIG. 10 illustrates an exemplary scheme for cell bead generation.

FIG. 11 illustrates an exemplary scheme for cell bead generation orfunctionalization using crosslinks.

FIGS. 12A-B illustrate exemplary scheme for polymerization orcrosslinking of polymer or gel precursors to generate cell beadscomprising cell bead index molecules.

FIGS. 13A-D illustrate an exemplary scheme for cell bead generation andfor the generation of partitions comprising cell beads and barcodebeads.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte or a cell type. A barcode can be part of an analyte. A barcodecan be independent of an analyte. A barcode can be a tag attached to ananalyte (e.g., nucleic acid molecule) or a cell type, or a combinationof the tag in addition to an endogenous characteristic of the analyte(e.g., size of the analyte or end sequence(s)) or cell type. A barcodemay be unique. A barcode may be identical, for example, when used toidentify a similar cell type. Barcodes can have a variety of differentformats. For example, barcodes can include: polynucleotide barcodes;random nucleic acid and/or amino acid sequences; and synthetic nucleicacid and/or amino acid sequences. A barcode can be attached to ananalyte in a reversible or irreversible manner. A barcode can be addedto, for example, a fragment of a deoxyribonucleic acid (DNA) orribonucleic acid (RNA) sample before, during, and/or after sequencing ofthe sample. A barcode can be added to, for example, a binding moietysuch as an antibody or antibody fragment. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. For example, the subject can be a vertebrate, a mammal, arodent (e.g., a mouse), a primate, a simian or a human. Animals mayinclude, but are not limited to, farm animals, sport animals, and pets.A subject can be a healthy or asymptomatic individual, an individualthat has or is suspected of having a disease (e.g., cancer) or apre-disposition to the disease, and/or an individual that is in need oftherapy or suspected of needing therapy. A subject can be a patient. Asubject can be a microorganism or microbe (e.g., bacteria, fungi,archaea, viruses).

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Polymers in the polymer matrix may be randomly arranged,such as in random copolymers, and/or have ordered structures, such as inblock copolymers. Cross-linking can be via covalent, ionic, orinductive, interactions, or physical entanglement. The bead may be amacromolecule. The bead may be formed of nucleic acid molecules boundtogether. The bead may be formed via covalent or non-covalent assemblyof molecules (e.g., macromolecules), such as monomers or polymers. Suchpolymers or monomers may be natural or synthetic. Such polymers ormonomers may be or include, for example, nucleic acid molecules (e.g.,DNA or RNA). The bead may be formed of a polymeric material. The beadmay be magnetic or non-magnetic. The bead may be rigid. The bead may beflexible and/or compressible. The bead may be disruptable ordissolvable. The bead may be a solid particle (e.g., a metal-basedparticle including but not limited to iron oxide, gold or silver)covered with a coating comprising one or more polymers. Such coating maybe disruptable or dissolvable.

The term “cell bead,” as used herein, generally refers to a hydrogel,polymeric, or crosslinked material that comprises (e.g., encapsulates,contains, etc.) a biological particle (e.g., a cell, a nucleus, a fixedcell, a cross-linked cell), a virus, components of, or macromolecularconstituents derived from a cell or virus. For example, a cell bead maycomprise a virus and/or a cell. In some cases, a cell bead comprises asingle cell. In some cases, a cell bead may comprise multiple cellsadhered together. A cell bead may include any type of cell, includingwithout limitation prokaryotic cells, eukaryotic cells, bacterial,fungal, plant, mammalian, or other animal cell types, mycoplasmas,normal tissue cells, tumor cells, a T-cell (e.g., CD4 T-cell, CD4 T-cellthat comprises a dormant copy of human immunodeficiency virus (HIV)), afixed cell, a cross-linked cell, a rare cell from a population of cells,or any other cell type, whether derived from single cell ormulticellular organisms. Furthermore, a cell bead may comprise a livecell, such as, for example, a cell may be capable of being cultured.Moreover, in some examples, a cell bead may comprise a derivative of acell, such as one or more components of the cell (e.g., an organelle, acell protein, a cellular nucleic acid, genomic nucleic acid, messengerribonucleic acid, a ribosome, a cellular enzyme, etc.). In someexamples, a cell bead may comprise material obtained from a biologicaltissue, such as, for example, obtained from a subject. In some cases,cells, viruses or macromolecular constituents thereof are encapsulatedwithin a cell bead. Encapsulation can be within a polymer or gel matrixthat forms a structural component of the cell bead. In some cases, acell bead is generated by fixing a cell in a fixation medium or bycross-linking elements of the cell, such as the cell membrane, the cellcytoskeleton, etc. In some cases, beads may or may not be generatedwithout encapsulation within a larger cell bead.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The sample may bea cell sample. The sample may be a cell line or cell culture sample. Thesample can include one or more cells. The sample can include one or moremicrobes. The biological sample may be a nucleic acid sample or proteinsample. The biological sample may also be a carbohydrate sample or alipid sample. The biological sample may be derived from another sample.The sample may be a tissue sample, such as a biopsy, core biopsy, needleaspirate, or fine needle aspirate. The sample may be a fluid sample,such as a blood sample, urine sample, or saliva sample. The sample maybe a skin sample. The sample may be a cheek swab. The sample may be aplasma or serum sample. The sample may be a cell-free or cell freesample. A cell-free sample may include extracellular polynucleotides.Extracellular polynucleotides may be isolated from a bodily sample thatmay be selected from the group consisting of blood, plasma, serum,urine, saliva, mucosal excretions, sputum, stool and tears.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample. Thebiological particle may be a virus. The biological particle may be acell or derivative of a cell. The biological particle may be anorganelle. The biological particle may be a rare cell from a populationof cells. The biological particle may be any type of cell, includingwithout limitation prokaryotic cells, eukaryotic cells, bacterial,fungal, plant, mammalian, or other animal cell type, mycoplasmas, normaltissue cells, tumor cells, or any other cell type, whether derived fromsingle cell or multicellular organisms. The biological particle may be aconstituent of a cell. The biological particle may be or may includeDNA, RNA, organelles, proteins, or any combination thereof. Thebiological particle may be or may include a matrix (e.g., a gel orpolymer matrix) comprising a cell or one or more constituents from acell (e.g., cell bead), such as DNA, RNA, organelles, proteins, or anycombination thereof, from the cell. The biological particle may beobtained from a tissue of a subject. The biological particle may be ahardened cell. Such hardened cell may or may not include a cell wall orcell membrane. The biological particle may include one or moreconstituents of a cell, but may not include other constituents of thecell. An example of such constituents is a nucleus or an organelle. Acell may be a live cell. The live cell may be capable of being cultured,for example, being cultured when enclosed in a gel or polymer matrix, orcultured when comprising a gel or polymer matrix.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from a biological particle. Themacromolecular constituent may comprise a nucleic acid. In some cases,the biological particle may be a macromolecule. The macromolecularconstituent may comprise DNA. The macromolecular constituent maycomprise RNA. The RNA may be coding or non-coding. The RNA may bemessenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), forexample. The RNA may be a transcript. The RNA may be small RNA that areless than 200 nucleic acid bases in length, or large RNA that aregreater than 200 nucleic acid bases in length. Small RNAs may include5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA(miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs),Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and smallrDNA-derived RNA (srRNA). The RNA may be double-stranded RNA orsingle-stranded RNA. The RNA may be circular RNA. The macromolecularconstituent may comprise a protein. The macromolecular constituent maycomprise a peptide. The macromolecular constituent may comprise apolypeptide.

The term “molecular tag” also referred to as “tag,” as used herein,generally refers to a molecule capable of binding to a macromolecularconstituent or a cellular marker on a surface of a cell. The moleculartag may bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode. The molecular tag can be coupled to apolynucleotide sequence to be “tagged” by any suitable approachincluding ligation, hybridization, or other approaches.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. A partition may be a physical compartment, suchas a droplet or well. The partition may isolate space or volume fromanother space or volume. The droplet may be a first phase (e.g., aqueousphase) in a second phase (e.g., oil) immiscible with the first phase.The droplet may be a first phase in a second phase that does not phaseseparate from the first phase, such as, for example, a capsule orliposome in an aqueous phase. A partition may comprise one or more other(inner) partitions. In some cases, a partition may be a virtualcompartment that can be defined and identified by an index (e.g.,indexed libraries) across multiple and/or remote physical compartments.For example, a physical compartment may comprise a plurality of virtualcompartments.

The term “microfluidic device,” as used herein generally refers to adevice configured for fluid transport and having a fluidic channelthrough which fluid can flow with at least one dimension of no greaterthan about 10 millimeters (mm). The dimension can be any of length,width or height. In some cases, a microfluidic device comprises afluidic channel having multiple dimensions of no greater than about 10mm. A microfluidic device can also include a plurality of fluidicchannels each having a dimension of no greater than about 10 mm. Thedimension(s) of a given fluidic channel of a microfluidic device mayvary depending, for example, on the particular configuration of thechannel and/or channels and other features also included in the device.

In some examples, a dimension of a fluidic channel of a microfluidicdevice may be at most about 10 mm, at most about 9 mm, at most about 8mm, at most about 7 mm, at most about 6 mm, at most about 5 mm, at mostabout 4 mm, at most about 3 mm, at most about 2 mm, at most about 1 mm,at most about 900 micrometers (μm), at most about 800 μm, at most 700μm, at most about 600 μm, at most about 500 μm, at most about 400 μm, atmost about 300 μm, at most about 200 μm, at most about 100 μm, at mostabout 90 μm, at most about 70 μm, at most about 60 μm, at most about 50μm, at most about 40 μm, at most about 30 μm, at most about 20 μm, atmost about 10 μm, at most about 8 μm, at most about 6 μm, at most about4 μm, at most about 2 μm, at most about 1 μm or less. In some examples adimension of a fluidic channel of a microfluidic device may be at leastabout 1 μm, at least about 2 μm, at least about 4 μm, at least about 6μm, at least about 8 μm, at least about 10 μm, at least about 20 μm, atleast about 30 μm, at least about 40 μm, at least about 50 μm, at leastabout 60 μm, at least about 70 μm, at least about 80 μm, at least about90 μm, at least about 100 μm, at least about 200 μm, at least about 300μm, at least about 400 μm, at least about 500 μm, at least about 600 μm,at least about 700 μm, at least about 800 μm, at least about 900 μm, atleast about 1 mm, at least about 2 mm, at least about 3 mm, at leastabout 4 mm, at least about 5 mm, at least about 6 mm, at least about 7mm, at least about 8 mm, at least about 9 mm, at least about 10 mm ormore.

Microfluidic devices described herein can also include any additionalcomponents that can, for example, aid in regulating fluid flow, such asa fluid flow regulator (e.g., a pump, a source of pressure, etc.),features that aid in preventing clogging of fluidic channels (e.g.,funnel features in channels; reservoirs positioned between channels,reservoirs that provide fluids to fluidic channels, etc.) and/orremoving debris from fluid streams, such as, for example, filters.Additional microfluidic features are described in U.S. PatentPublication No. 2015/0292988, which is herein incorporated by referencein its entirety. Moreover, microfluidic devices may be configured as afluidic chip that includes one or more reservoirs that supply fluids toan arrangement of microfluidic channels and also includes one or morereservoirs that receive fluids that have passed through the microfluidicdevice. In addition, microfluidic devices may be constructed of anysuitable material(s), including polymer species and glass.

Indexing of Cells for Multiplex Analysis

Disclosed herein, in some embodiments, are methods for cellularanalysis, comprising (a) providing a first cell bead comprising (i) afirst biological particle; and (ii) a nucleic acid molecule comprising afirst cell bead index sequence; (b) providing a second cell beadcomprising (i) a second biological particle; and (ii) a nucleic acidmolecule comprising a second cell bead index sequence different thansaid first cell bead index sequence; (c) pooling said first cell beadand said second cell bead to provide a pooled plurality of cell beads;and (d) partitioning said pooled plurality of cell beads into aplurality of partitions to generate (i) a first partition comprisingsaid first cell bead and a nucleic acid molecule comprising a firstbarcode sequence and (ii) a second partition comprising said second cellbead and a nucleic acid molecule comprising a second barcode sequence,wherein said second barcode sequence is different than said firstbarcode sequence. In some instances, the methods disclosed hereinfurther comprise: (e) generating (i) a first nucleic acid moleculecomprising said first cell bead index sequence and said first barcodesequence and (ii) a second nucleic acid molecule comprising said secondcell bead index sequence and said second barcode sequence.

Also disclosed herein, in some embodiments, are compositions comprising:(a) a first cell bead comprising (i) a first biological particle; and(ii) a nucleic acid molecule comprising a first cell bead indexsequence; and (b) a second cell bead comprising (i) a second biologicalparticle; and (ii) a nucleic acid molecule comprising a second cell beadindex sequence different than said first cell bead index sequence.

In an aspect, the compositions, methods, and systems described hereinfacilitate the identification and/or sample multiplexing of cell beadsfrom one or more cell bead samples. Cell bead samples can include, butare not limited to, cell beads comprising a biological particle from orderived from different cell types (including cells from different tissuetypes of an individual or the same tissue type from differentindividuals) or different biological organisms such as microorganismsfrom differing genera, species, strains, variants, or any combinationthereof. In some instances, the microorganism can be a bacterium, anarchaeon, a fungus, a virus, a protozoan, or an alga. The cell samplesmay originate from cells with a similar or identical genetic background.The cell type can be a human cell. Differing cell types may include, forexample, normal and tumor tissue from an individual, various cell typesobtained from a human subject such as a variety of immune cells (e.g., Bcells, T cells, and the like), multiple different bacterial species,strains and/or variants from environmental, forensic, microbiome orother samples, or any of a variety of other mixtures of cell types. Insome cases, the methods and systems described herein comprise combiningcells from two, three, four, five, six, seven, eight, nine, ten, or morethan ten cell types or cell populations into one multiplexed assay. Cellbead samples can also comprise discrete sets of cell beads comprising orderived from a common type of biological particle (e.g., a plurality ofcell beads derived from the same cell type). For example, a first cellbead sample and a second cell bead sample can each comprise or bederived from a common biological particle (e.g., a common cell type) butare processed or otherwise maintained as separate populations. Forinstance, the first and second cell bead samples can be subjected todiffering experimental conditions or treatments. In other instances, thefirst and second cell bead samples can be technical replicates.

For example, in some embodiments, a cell bead is generated as describedelsewhere herein (e.g., using a microfluidic device), the cell beadcomprising: (i) a biological particle (e.g., a cell or a derivative of acell); and (ii) a nucleic acid molecule comprising a cell bead indexsequence. In some instances, a plurality of cell beads is generated asdescribed elsewhere herein, wherein each cell bead of the plurality ofcell beads comprises (i) a biological particle (e.g., a cell or aderivative of a cell); and (ii) a nucleic acid molecule comprising acommon cell bead index sequence. As such, each cell bead comprising thecommon cell bead index sequence can be identified, detected, orotherwise associated with the plurality of cell beads. Thus, using thecompositions, systems, and methods described herein, cell beads fromdifferent samples (e.g., different cells/biological particles, cellpopulations, cell types, experimental conditions, etc.) can be “tagged”with different cell bead index sequences and subsequently combined formultiplex processing and analysis. In other words, because each cellbead generated from a sample can comprise a common cell bead indexsequence, and because each cell bead sample can comprise a unique cellbead index sequence, multiple and/or different samples can be combinedinto a single assay thereby increasing throughput and saving time andresources associated with downstream processing of discrete samples.

Cell beads comprising a cell bead index molecule comprising a cell beadindex sequence can be generated using any suitable method(s) describedherein. For a description of cell beads and cell bead generationstrategies, see, e.g., U.S. Pat. Pub. US 2018/0216162, PCT ApplicationPublication No. WO2019071039, filed Oct. 4, 2018, and U.S. Pat. Pub.20190100632, all of which are hereby incorporated by reference in theirentirety. For example, in some embodiments, a biological particle (e.g.,a cell or cell nucleus) is partitioned into a partition (e.g., a dropletin an emulsion) with polymeric or gel precursors and a nucleic acidmolecule comprising a cell bead index sequence. In some instances, abiological particle (e.g., a single biological particle, such as asingle cell) is partitioned into a partition with polymeric or gelprecursors and a plurality of nucleic acid molecules each comprising acommon cell bead index sequence. The partition is subjected toconditions sufficient to polymerize or cross-link the polymeric or gelprecursors to generate the cell bead, wherein the cell bead encapsulatesthe biological particle and the cell bead index molecule(s).

In some cases, cell beads can be synthesized in one-step procedures,e.g., polymerization and concurrent cross-linking reactions ofmultifunctional monomers. In other cases, cell beads can be synthesizedin multi-steps procedures, e.g., polymerization of monomers first,followed by crosslinking reactions by using, e.g., orthogonal, reactivegroups that can respond to different conditions to allow stepwiseapproaches.

Cell beads can be synthesized by techniques that can create acrosslinked polymer. In some cases, copolymerization/cross-linking freeradical polymerizations can be used to produce hydrogels by reactinghydrophilic monomers with multifunctional crosslinking molecules. Thiscan be done by, for example, linking polymer chains via a chemicalreaction(s), using ionizing radiation to generate main-chain freeradicals which can recombine as crosslinking junctions, or physicalinteractions such as entanglements, electrostatics, and crystalliteformation. Types of polymerization can include bulk, solution, andsuspension polymerization.

Suspension polymerization or dispersion polymerization can be employedin water-in-oil or emulsion processes, sometimes called “inversionsuspension.” In some cases, the monomers and initiators can be dispersedin the oil or hydrocarbon phase as a homogenous mixture. In some cases,two types of polymer molecules can be first produced, each having areactive, crosslinking moiety for cross-linking purposes. Then these twotypes of polymer molecules can be enclosed in an emulsion such that thetwo reactive, crosslinking moieties can react and form crosslinksbetween the two types of polymers, thereby completing the synthesis ofthe hydrogel.

In some cases, cell beads can be synthesized from monomers,polymerization initiators, and crosslinking reagents. After thepolymerization reactions are complete, the hydrogels formed can beseparated from remaining starting materials and unwanted by-products,etc. The length of the polymer formed can be controlled depending on thedesired properties of the hydrogels.

Types of polymerizations employed to synthesize hydrogels can include,but are not limited to, free radical polymerization, controlled radicalpolymerization, crosslinking polymerization, networks formation ofwater-soluble polymers, and radiation crosslinking polymerization, etc.Polymerization can be initiated by initiators or free-radical generatingcompounds, such as, for example, benzoyl peroxide,2,2-azo-isobutyronitrile (AIBN), and ammonium peroxodisulphate, or byusing UV-, gamma- or electron beam-radiation.

For example, as shown in FIG. 10 , cells and polymer or gel precursorsare mixed with an immiscible fluid (e.g., an oil), thereby generating aplurality of aqueous droplets, including droplet 1001 comprising abiological particle, in this instance a cell 1002. Droplet 1001 may alsocomprise a cell bead index molecule 1005, as described herein. Droplet1001 is subjected to conditions sufficient for polymerization orgelation of the polymer or gel precursors to generate a cell bead 1003comprising cell 1002 and cell bead index molecule(s) 1005. Gelation maycomprise any of the gelation mechanisms and polymers described herein.In some instances, cell bead 1003 is subjected to treatment conditionssufficient to lyse cell 1002, releasing components of the cell into thecell bead. In other embodiments, cell 1002 is lysed in droplet 1001prior to polymerization or gelation of the polymer or gel precursors togenerate cell bead 1003 comprising cell bead index molecule(s) 1005. Instill other embodiments, cell 1002 is permeabilized before, during, orafter polymerization or gelation of the polymer or gel precursors. Cellbeads are collected to generate a plurality of cell beads 1004. Cellbeads may be stored for further processing. In some cases, cell beadindex molecule(s) 1005 may be attached to the cell beads subsequent topolymerization or gelation of the polymer or gel precursor. Forinstance, polymer or gel precursors may comprise one or more functionalgroups that facilitate the attachment of the cell bead index molecule(s)1005 subsequent to polymerization or gelation of the polymer or gelprecursors. In other embodiments, the polymer or gel precursors and/orcell bead index molecule(s) 1005 comprise functional groups, whichfacilitate the incorporation of cell bead index molecule(s) 1005 intothe cell bead during polymerization or gelation of the polymer or gelprecursors.

In some embodiments, the cell bead index molecule(s) are entrappedwithin the cell bead polymeric and/or crosslinked matrix (also referredto herein as a “cell bead matrix”). In other embodiments, the cell beadindex molecule(s) are functionalized with chemical groups (e.g.,acrydite, amine, thiol, etc.) such that the cell bead index molecule(s)are incorporated into or otherwise attached to the cell bead matrix. Forexample, in a cell bead matrix comprising polyacrylamide, the cell beadindex molecule(s) can comprise an acrydite moiety such that, uponpolymerization of acrylamide monomers, the cell bead index molecule(s)are incorporated into the cell bead matrix. In some embodiments, boththe cell bead index molecule(s) and/or the cell bead matrix comprise oneor more functional groups configured to facilitate attachment of thecell bead index molecule(s) to the cell bead matrix. For example, insome embodiments, generation of a cell bead comprising a cell bead indexmolecule comprises: (a) providing a polymer or gel precursor (e.g., in apartition), wherein the polymer or gel precursor comprises a pluralityof first crosslink precursors; (b) providing a cell bead index moleculecomprising a second crosslink precursor; and (c) crosslinking thepolymer or gel precursor and the cell bead index molecule via a reactionbetween a first section of the first crosslink precursors and a secondsection of the second crosslink precursors, thereby forming the cellbead comprising the cell bead index molecule.

In some instances, the cell bead index molecule(s) are irreversiblyincorporated into the cell bead matrix. In other instances, the cellbead index molecule(s) are reversibly incorporated into the cell beadmatrix. For example, a cell bead index molecule(s) can be functionalizedwith a labile moiety as described elsewhere herein (e.g., a disulfidebond) such that the cell bead index molecule, or a portion thereof, isconfigured to be released from the cell bead matrix and/or cell bead.

In some embodiments, the cell bead matrix includes one or more of thefollowing; disulfide crosslinked polyacrylamide, agarose, alginate,polyvinyl alcohol, PEG-diacrylate, PEG-acrylate/thiol, PEG-azide/alkyne,other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin,elastin, a polyolefin, an olefin copolymers, an acrylics, a vinylpolymer, a polyesters, a polycarbonate, a polyamide, a polyimide, aformaldehyde resin, a polyurethane, an ether polymer, a cellulosic, athermoplastic elastomer, a thermoplastic polyurethane, or any polymericprecursor (e.g., monomer) thereof. In some embodiments, the cell beadmatrix comprises polyacrylamide (e.g., disulfide crosslinkedpolyacrylamide).

In some embodiments, generation of the cell bead matrix comprises (a)providing a first polymer or gel precursor, wherein the first polymer orgel precursor comprises a plurality of first crosslink precursors, forexample a moiety comprising an azide group; (b) providing a secondpolymer or gel precursor, wherein the second polymer or gel precursorcomprises a plurality of second crosslink precursors, for example amoiety comprising an alkyne group; and (c) crosslinking the firstpolymer and the second polymer via a reaction (e.g., a click-chemistryreaction) between a first section of the first crosslink precursors anda second section of the second crosslink precursors, thereby forming thecell bead.

For example, as shown in FIG. 11 , emulsion systems 1100, 1102, and 1104represent different stages through which polymer molecules or gelprecursors are crosslinked to form a cell bead matrix or hydrogel.Emulsion system 1100 can comprise a discrete droplet 1108 (comprising anaqueous phase) immersed in an oil phase 1110. Within the discretedroplet 1108, two polymer molecules 1112 and 1114 and a biologicalparticle (e.g., a single biological particle, such as a single cell ornucleus—not shown) can be partitioned together. In some instances, acell bead index molecule(s) (not shown) is also partitioned with thepolymer molecules or gel precursors and the biological particle. In someembodiments, the cell bead index molecule(s) comprise a functional group(e.g., a click chemistry moiety such as 1118 or 1120) to facilitateattachment to the cell bead matrix. Polymer molecule 1112 can comprise afirst crosslink precursor comprising a first click chemistry moiety 1118and optionally a labile bond 1116 (e.g., a chemically, thermally,enzymatically, or photo-labile bond). Polymer molecule 1114 can comprisea second click chemistry moiety 1120. In the oil phase 1110, there canbe other reagents, such as reagent 1122 (shown as a copper (II)reagent), which may be utilized to facilitate the click chemistryreaction between the first click chemistry moiety 1118 and the secondclick chemistry moiety 1120, either by itself or by a derivativethereof. Because the reagent 1122 remains outside of the discretedroplet 1108, generally no click chemistry reaction happens within thediscrete droplet 1108 in the absence of the reagent 1122.

In emulsion system 1102, some of the reagent 1122 can penetrate thediscrete droplet 1108, via, e.g., physical or chemical processes. Insome instances, reagent 1122 becomes or is otherwise processed to becomereagent 1124 (shown as a copper (I) reagent) in the discrete droplet1108. In some instances, conversion into reagent 1124 requiresadditional reagents (not shown, e.g., a reducing agent such as sodiumascorbate). In these embodiments, reagent 1124 can be the reagentrequired to initiate the click chemistry reaction between the firstclick chemistry moiety 1118 and the second click chemistry moiety 1120.Once in the proximity of both the first click chemistry moiety 1118 andthe second click chemistry moiety 1120, the reagent 1124 can initiate aclick chemistry reaction, such as a Cu(I)—Catalyzed Azide-AlkyneCycloaddition (CuAAC), see emulsion system 1104. In embodiments wherethe cell bead index molecules comprise a click-chemistry moiety, thereagent can also catalyze the attachment of cell bead index molecules tothe cell bead matrix.

As shown in the emulsion system 1104 of FIG. 11 , in the presence of thereagent 1124, a crosslink 1126 is formed linking the two polymermolecules 1112 and 1114 together, via the newly formed moiety 1128because of the click chemistry reaction between the first clickchemistry moiety 1118 and the second click chemistry moiety 1120. Ahydrogel comprising the crosslinked polymer molecules 1112 and 1114 canthus be formed, thereby generating the cell bead. Reagents 1122 and/or1124 can be removed from the newly formed hydrogel if desired. In someinstances, the cell bead matrix comprises a labile bond 1116 (e.g., adisulfide bond) configured to release the crosslinks 1126 and/or degradethe hydrogel upon application of a stimulus (e.g., a chemical, thermal,or photo-stimulus). In some instances, the cell bead index molecules areattached to the hydrogel via a labile bond 1116 configured to releasethe cell bead index molecules from the cell bead matrix.

In some embodiments, the cell bead index molecule(s) described hereinare attached, entrapped, or otherwise incorporated into the cell beadmatrix during cell bead generation (see, e.g., FIG. 10 and FIG. 11 ). Inother embodiments, the cell bead index molecule(s) described herein areattached, entrapped, or otherwise incorporated into the cell bead matrixsubsequent to cell bead generation. For example, in some instances, acell bead can be generated as described elsewhere herein and a cell beadindex molecule can be attached to the cell bead matrix by a chemicalreaction, e.g., between a functional group of the cell bead indexmolecule(s) and a functional group in the cell bead matrix.

FIGS. 12A-B illustrates an example of generating cell beads comprisingcell bead index molecule(s) 1202 attached to a polymer matrix. Forinstance, as shown in FIG. 12A, a partition 1200 comprising gel orpolymer precursors 1201 attached to cell bead index molecule(s) 1202 canbe subjected to conditions sufficient to polymerize, gel, or crosslinkthe precursors 1201, thereby generating a cell bead 1210 comprising cellbead index molecule(s) 1202 attached to the polymer matrix 1203. In someinstances, a partition 1220 comprising a first polymer or gel precursor1201 attached to cell bead index molecule(s) 1202 and a second polymeror gel precursor 1204 can be subjected to conditions sufficient topolymerize, gel, or crosslink precursors 1201 and 1204, therebygenerating a cell bead 1230 comprising cell bead index molecule(s) 1202attached to a polymer 1205 of polymer or gel precursors 1201 and 1204.In some instances, polymer or gel precursor 1201 is a first type ofpolymer, polymer or gel precursor 1204 is a second type of polymer, andpolymer 1205 is a copolymer of precursors 1201 and 1204. In otherinstances, polymer or gel precursor 1201 is a first type of polymercomprising a cell bead index molecule 1202 and polymer or gel precursor1204 is the same type of polymer as 1201 but lacks the cell bead indexmolecule 1202.

In other embodiments, as shown in FIG. 12B, a partition 1240 is providedcomprising gel or polymer precursors 1201 comprising a first crosslinkprecursor 1206 (e.g., a first click chemistry moiety) and a cell beadindex molecule 1202 comprising a second crosslink precursor 1207 (e.g.,a second click chemistry moiety), wherein the first crosslink precursor1206 and the second crosslink precursor 1207 are configured to form acrosslink 1209 thereby linking the cell bead index molecule 1202 withthe polymer or gel precursor 1201 or with a polymerized gelled, orotherwise crosslinked matrix of 1201 (e.g., 1211).

In some instances, a partition 1260 is provided comprising (i) a firstpolymer or gel precursor 1201 comprising a first crosslink precursor1206 (e.g., a first click chemistry moiety), (ii) a second polymer orgel precursor 1204, and (iii) a cell bead index molecule 1202 comprisinga second crosslink precursor 1207 (e.g., a second click chemistrymoiety), wherein the first crosslink precursor 1206 and the secondcrosslink precursor 1207 are configured to form a crosslink 1209 therebylinking the cell bead index molecule 1202 with the polymer or gelprecursor 1201 or with a polymerized gelled, or otherwise crosslinkedmatrix of 1201 and 1212 (e.g., 1213). In some instances, a partition1260 comprising the first polymer or gel precursor 1201 attached to cellbead index molecule(s) 1202 and the second polymer or gel precursor 1212are subjected to conditions sufficient to polymerize, gel, or crosslinkprecursors 1201 and 1212, thereby generating a cell bead 1270 comprisingcell bead index molecule(s) 1202 attached to a polymer or gel 1213 ofpolymer or gel precursors 1201 and 1212. In some instances, polymer orgel precursor 1201 is a first type of polymer, polymer or gel precursor1212 is a second type of polymer, and polymer 1213 is a copolymer ofprecursors 1201 and 1212. In other instances, polymer or gel precursor1201 is a first type of polymer comprising a cell bead index molecule1202 and polymer or gel precursor 1212 is the same type of polymer as1201 but lacks the cell bead index molecule 1202.

In some instances, one or more agents are utilized to catalyze,initiate, or otherwise facilitate the formation of crosslink 1209. Insome instances, the partition 1240 is subjected to conditions sufficientto form a crosslink 1209 between crosslink precursors 1206 and 1209prior to polymerization, gelling, or crosslinking of polymer precursors(e.g., 1201 and/or 1212) to form cell bead 1250 or 1270. In otherinstances, the partition (e.g., 1240 or 1260) is subjected to conditionssufficient to form a crosslink 1209 between crosslink precursors 1206and 1209 concurrently with the polymerization, gelling, or crosslinkingof the polymer or gel precursors (e.g., 1201 and/or 1212). In someembodiments, the partition (e.g., 1240 or 1260) is subjected toconditions sufficient to polymerize, gel, or otherwise crosslink thepolymer or gel precursors (e.g., 1201 and/or 1212) prior to forming acrosslink 1209 between crosslink precursors 1206 and 1209. In someinstances, the cell bead index molecule comprises a labile bond 1208configured to release the crosslink 1209 and the cell bead indexmolecule 1202 upon application of a stimulus (e.g., a chemical, thermal,or photo-stimulus).

In some instances, a cell bead index molecule(s) 1202 is attached to thefirst polymer or gel precursor (e.g., 1201), the second polymer or gelprecursors (e.g., 1204 or 1212), or both the first 1201 and the secondpolymer or gel precursors (e.g., 1204 or 1212). Furthermore, in someembodiments, additional polymers or polymer or gel precursors can beadded (e.g., to partition 1200, 1220, 1240, or 1260) to generate aco-polymer or mixed polymer cell bead matrix. Additionally, theconcentration of polymers (e.g., 1201, 1204, and/or 1212) in thepartition (e.g., 1200, 1220, 1240, or 1260) can be controlled togenerate a cell bead comprising a desired concentration of cell beadindex molecules 1202.

In some cases, the cell beads disclosed herein can comprise polymerssuch as poly(acrylic acid), poly(vinyl alcohol), poly(vinylpyrrolidone),poly(ethylene glycol), polyacrylamide, some polysaccharides, or anyderivatives thereof. These polymers can be non-toxic and they can beused in various pharmaceutical and biomedical applications. Thus, insome instances, they may not require their removal from the reactionsystem, thereby eliminating the need for a purification step after theformation of hydrogels.

Polymers can comprise polymer molecules of a particular length or rangeof lengths. Polymer molecules can have a length of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 1,000,2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000,1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000, 100,000,000,200,000,000, 500,000,000 or 1,000,000,000 backbone atoms or molecules(e.g., carbons). Polymer molecules can have a length of at most 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000,500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000,100,000,000, 200,000,000, 500,000,000 or 1,000,000,000 backbone atoms ormolecules (e.g., carbons). Polymer molecules can have a length of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000,100,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000,20,000,000, 100,000,000, 200,000,000, 500,000,000 or 1,000,000,000monomer units (e.g., vinyl molecules or acrylamide molecules). Polymermolecules can have a length of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 1,000, 2,000,5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000,2,000,000, 5,000,000, 10,000,000, 20,000,000, 100,000,000, 200,000,000,500,000,000 or 1,000,000,000 monomer units (e.g., vinyl molecules oracrylamide molecules).

In some embodiments, the cell bead index molecule(s) described hereinare single-stranded nucleic acid molecules. In some embodiments, thecell bead index molecule(s) described herein are double-stranded nucleicacid molecules. In some embodiments, the cell bead index molecule(s)described herein are partially double-stranded nucleic acid molecules.In some embodiments, the cell bead index molecules described hereincomprise a cell bead index sequence (i.e., a “barcode” as describedelsewhere herein). Cell bead index molecules can also compriseadditional functional sequences that facilitate one or more nucleic acidreactions. For example, in some instances, a cell bead index moleculecomprises a cell bead index sequence and one or more of a primer orprimer binding sequence (e.g., a sequencing primer or primer bindingsequence, such as an R1 or R2 sequence, or a partial sequence thereof),a sequence configured to attach to a flow cell of a sequencer (e.g., aP5 or P7 sequence, or a partial sequence thereof), a unique molecularindex sequence (UMI), or any other suitable functional sequencedescribed herein. In some embodiments, the cell bead index moleculesdescribed herein comprise a sequence configured to couple or hybridizeto a complementary (or partially complementary) sequence on anothernucleic acid molecule (e.g., a nucleic acid barcode molecule). Forexample, in some cases, a cell bead index molecule comprises a cell beadindex sequence and a poly-A sequence configured to couple or hybridizeto a poly-T sequence on another nucleic acid molecule. In other cases, acell bead index molecule comprises a cell bead index sequence and asequence configured to couple or hybridize to a template switchingoligonucleotide (TSO) or TSO sequence on another nucleic acid molecule(e.g., a nucleic acid barcode molecule, such as a nucleic acid barcodemolecule attached to a bead). For a description of exemplary nucleicacid barcode molecule configurations, see, e.g., U.S. Pat. Pub.20150376609, which is hereby incorporated by reference in its entirety.

In some cases, the length of a cell bead index molecule is 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249 or 250 nucleotides or longer.

In some cases, the length of a cell bead index molecule is at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 nucleotides orlonger.

In some cases, the length of a cell bead index molecule is at most 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 nucleotides.

Partitioning Cell Beads

Cell beads comprising cell bead index molecules may be partitionedtogether with nucleic acid barcode molecules and the nucleic acidmolecules of or derived from the biological particle of the cell bead(e.g., mRNA, cDNA, gDNA, etc) and the cell bead index molecules can bebarcoded as described elsewhere herein. In some embodiments, cell beadscomprising cell bead index molecule(s) are co-partitioned (e.g., in awell or droplet emulsion) with barcode carrying beads (e.g., gel beads)and the nucleic acid molecules of or derived from the cell bead and thecell bead index molecules are barcoded as described elsewhere herein. Anoverview of an exemplary method for generating partitions comprisingcell beads and nucleic acid barcode molecules is schematically depictedin FIG. 13A. The method described in FIG. 13A comprises three phases1310, 1320, and 1330 with each respective phase comprising: (1)generation of cell beads (1310); (2) cell bead solvent exchange andoptional processing (1320); and (3) co-partitioning of cell beads andbarcodes for subsequent tagging (e.g., barcoding) of one or moreconstituents of (or derived from) the cell bead and the cell bead indexmolecule(s) (1330).

With continued reference to FIG. 13A, phase 1310 comprises providing anoil 1301, polymeric or gel precursors 1302, and biological particles1303 (e.g., a cell, a fixed cell, a cross-linked cell, a nucleus, apermeabilized nuclei, etc.) to a microfluidic chip (e.g., 1304) fordroplet generation. Cell bead index molecules, such as those describedelsewhere herein, may be further provided to microfluidic chip 1304 forco-partitioning. In some instances, the cell bead index molecules areprovided with or otherwise attached to the polymeric or gel precursors1302. In other cases, the cell bead index molecules are provided withthe biological particles 1303. As detailed in FIG. 13B, in someinstances, the microfluidic chip 1304 comprises a plurality ofmicrofluidic channels 1304 a connected to a plurality of reservoirscomprising the oil 1301, polymeric or gel precursors 1302, andbiological particles (e.g., cells) 1303. Microfluidic chip 1304 may alsocomprise one or more additional channels and/or reservoirs (not shown)comprising one or more additional reagents (such as the cell bead indexmolecules described herein). Other suitable microfluidic chiparchitectures (e.g., FIGS. 4-7 ) can also be utilized with the methodsand systems disclosed herein. Polymeric or gel precursors 1302 andbiological particles 1303 (and in some cases, cell bead index molecules)are flowed (e.g., via the action of an applied force, such as negativepressure via a vacuum or positive pressure via a pump) from theirreservoirs through the plurality of microfluidic channels (e.g., 1304 a;see also FIGS. 4-7 ) to a first channel junction and combine to form anaqueous stream. This aqueous stream is then flowed to a second channeljunction, in which oil 1301 is provided. The aqueous stream providedfrom the first channel junction is immiscible with the oil 1301resulting in the generation of a suspension of aqueous droplets 1305 inthe oil, which then flow to a reservoir for collection 1307. Flow can becontrolled within the microfluidic chip 1304 via any suitable method,including the use of one or more flow regulators in a channel or variouschannels, dimensioning of microfluidic channels, etc., as describedelsewhere herein. As shown in FIG. 13A, the product comprises droplets1305 comprising a biological particle 1303, the polymeric or gelprecursors 1302, and in some cases, a cell bead index molecule(s). Insome cases, at least some of the droplets of droplets 1305 comprise asingle biological particle (e.g., a single cell or single nucleus).

In some embodiments, the droplets 1305 are subjected to conditionssufficient to lyse the biological particles (e.g., cells or nuclei)comprised therein, releasing cellular macromolecular constituents intothe droplets 1305. The macromolecular constituents (e.g., nucleic acids,proteins, etc.) may additionally be subjected to one or more reactionsfor processing as described elsewhere herein. In other embodiments, thedroplets 1305 are subjected to conditions sufficient to permeabilize thecells (or nuclei) thereby facilitating access to one or moremacromolecular constituents of the cell (or nucleus) for furtherprocessing. In still other cases, the biological particles present inthe droplets 1305 are not lysed or permeabilized.

Continuing with FIG. 13A, the droplets 1305 comprising biologicalparticles are then subjected to conditions suitable to polymerize or gelthe polymeric or gel precursors 1302 in the droplets 1305, to generatecell beads 1306. In instances where cell bead index molecules areincluded during cell bead generation, cell beads 1306 also comprise cellbead index molecules. As the resulting cell beads 1306 are suspended inoil, in some embodiments, phase 1320 is initiated which comprises asolvent exchange configured to resuspend the cell beads 1306 in anaqueous phase 1311.

In some embodiments, the resuspended aqueous cell beads 1311 areoptionally processed to, e.g., prepare the cell beads for analysis ofone or more cellular components. For example, cell beads 1311 can besubjected conditions suitable to lyse or permeabilize biologicalparticles (e.g., cells or nuclei) in the cell beads 1313, therebyreleasing or otherwise allowing access to one or more cellularconstituents (e.g., nucleic acids, such as mRNA and gDNA, proteins,etc.). Separately or contemporaneously from cell lysis, cell beads(e.g., 1311 or 1313) are also subjected to conditions sufficient todenature nucleic acids derived from the cells (e.g., gDNA) associatedwith the cell beads (e.g., using NaOH). The polymeric matrix of the cellbeads (e.g., 1311 or 1313) effectively hinders or prohibits diffusion oflarger molecules, such as nucleic acids and/or proteins, from the cellbeads, but are sufficiently porous to facilitate diffusion ofdenaturation or other agents into the cell bead matrix to contactnucleic acids and other cellular components within the cell beads. Insome cases, the cell beads (1311 or 1313) can be subjected to conditionssuitable for performing one or more reactions on nucleic acids or otheranalytes derived from the cells associated with the cell beads (1311 or1313). For example, antibodies may be washed into and/or out of theresuspended cell beads (1311 or 1313). Additionally, in embodimentswhere cell bead index molecules are attached or otherwise incorporatedinto the cell beads after cell bead generation, one or more reactionscan be performed on the cell bead (1311 or 1313) to attach or otherwiseincorporate the cell bead index molecules into the cell beads (e.g.,through functional groups on the cell bead index molecule(s), cell beadmatrix, or both).

In some instances, one or more analytes of a cell (e.g., cell surfacecomponents, such as a protein) can be analyzed using polynucleotideconjugated labelling agents as described e.g., in U.S. Pat. Nos.9,951,386; 10,480,029; and U.S. Pat. Pub. 20190367969 the disclosures ofwhich are incorporated by reference herein in their entirety. Briefly, alibrary of potential labelling agents may be provided associated with afirst set of nucleic acid reporter molecules, e.g., where a differentreporter oligonucleotide sequence is associated with a specificlabelling agent, and therefore capable of binding to a specific cellanalyze (such as a cell surface feature or intracellular analyte such asa protein). Labelling agents may include, but are not limited to, anantibody, or an epitope binding fragment thereof, a cell surfacereceptor binding molecule, a receptor ligand, a small molecule, abi-specific antibody, a bi-specific T-cell engager, a T-cell receptorengager, a B-cell receptor engager, a pro-body, an aptamer, a monobody,an affimer, a darpin, and a protein scaffold. In some aspects, differentmembers of the library may be characterized by the presence of adifferent oligonucleotide sequence label, e.g., an antibody to a firsttype of cell surface protein or receptor may have associated with it afirst known reporter oligonucleotide sequence, while an antibody to asecond cell surface protein or receptor may have a different knownreporter oligonucleotide sequence associated with it. Prior toco-partitioning, the indexed cell beads may be incubated with thelibrary of labelling agents, that may represent antibodies to a broadpanel of different cell surface features, e.g., receptors, proteins,etc., and which include their associated reporter oligonucleotides.Unbound labelling agents may be washed from the cells, and the cells maythen be co-partitioned along with the barcode oligonucleotides describedabove. As a result, the partitions may include the indexed cell beads,as well as the bound labelling agents and their known, associatedreporter oligonucleotides. Labelling agent reporter oligonucleotides canthen be barcoded, optionally further processed, and sequenced asdescribed generally herein.

In some instances, open chromatin (e.g., ATAC-seq) or genomic DNAmethylation status is interrogated as described, e.g., in U.S. Pat. Pub.20180340171 and U.S. Pat. Pub. 20190367969, the disclosures of which arehereby incorporated by reference in their entirety.

After optional processing, the cell beads comprising the cell bead indexmolecules can be collected and stored prior to initiation of phase 1330.

In some embodiments, a plurality of first cell beads are generated froma first plurality of biological particles and a plurality of second cellbeads are generated from a second plurality of biological particles. Forexample, in FIG. 13C, a first plurality of biological particles (e.g.,cells) is loaded into a first reservoir (e.g., 1331) of a microfluidicdevice (e.g., 1304) and a second plurality of biological particles(e.g., cells) is loaded into a second reservoir (e.g., 1332) of themicrofluidic device. A plurality of first cell beads derived from thefirst plurality of biological particles and a plurality of second cellbeads derived from the second plurality of biological particles are thengenerated (e.g., see phase 1310 described above). In some instances, aplurality of cell bead index molecules, each comprising a cell beadindex sequence, are also added to the microfluidic device to generate aplurality of first cell beads comprising a plurality of first cell beadindex molecules comprising a first cell bead index sequence and aplurality of second cell beads comprising a plurality of second cellbead index molecules comprising a second cell bead index sequence. Insome embodiments, a third plurality of biological particles is loadedinto a third reservoir (e.g., 1333) of the microfluidic device (e.g.,1304) to generate a third plurality of cell beads comprising a thirdcell bead index sequence. In some embodiments, a fourth plurality ofbiological particles is loaded into a fourth reservoir (e.g., 1334) ofthe microfluidic device (e.g. 1304) to generate a fourth plurality ofcell beads comprising a fourth cell bead index sequence. In someembodiments, a fifth plurality of biological particles is loaded into afifth reservoir (e.g., 1335) of the microfluidic device (e.g., 1304) togenerate a fifth plurality of cell beads comprising a fifth cell beadindex sequence. In some embodiments, a sixth plurality of biologicalparticles is loaded into a sixth reservoir (e.g., 1336) of themicrofluidic device (e.g., 1304) to generate a sixth plurality of cellbeads comprising a sixth cell bead index sequence. In some embodiments,a seventh plurality of biological particles is loaded into a seventhreservoir (e.g., 1337) of the microfluidic device (e.g., 1304) togenerate a seventh plurality of cell beads comprising a seventh cellbead index sequence. In some embodiments, an eighth plurality ofbiological particles is loaded into an eighth reservoir (e.g., 1338) ofthe microfluidic device (e.g., 1304) to generate an eighth plurality ofcell beads comprising an eighth cell bead index sequence. Although themicrofluidic device 1304 shown in FIGS. 13B-13C can generate up to 8discrete cell bead samples (e.g., 8 pluralities of cell beads, eachplurality of cell beads comprising a common cell bead index molecule),any suitable microfluidic device comprising any suitable number ofchannels, reservoirs, or microfluidic configurations can be utilizedwith the compositions and methods disclosed herein. Likewise, becausedifferent cell bead index sequences can be utilized to distinguishingdiscrete cell bead samples, any suitable number of uniquely labeled cellbead samples can be generated using the methods and compositionsdescribed herein.

After generation of cell beads comprising cell bead index molecules,cell bead samples can be pooled together for subsequent characterizationand analysis. For example, as shown in FIG. 13C, in some embodiments,droplets 1305 comprising biological particles and cell bead indexmolecules are collected from each reservoir 1307 and combined forfurther processing (e.g., gelation/polymerization, solvent exchange,cell lysis, etc.) in bulk to generate a pooled plurality of cell beadscomprising cell bead index molecules 1314. In other embodiments,droplets 1305 comprising biological particles and cell bead indexmolecules are collected from each reservoir 1307 and processedseparately in one or more reactions (e.g., gelation/polymerization,solvent exchange, cell lysis, etc.) and subsequently combined togenerate a pooled plurality of cell beads comprising cell bead indexmolecules 1314. Because each cell bead of the pooled plurality of cellsbeads 1314 comprises a cell bead index molecule comprising a cell beadindex sequence, the sample of origin of each cell bead of the pooledplurality of cell beads can be determined.

Continuing with FIG. 13A, after phase 1320, cell beads from the pooledplurality of cell beads 1314 can be analyzed by, e.g., partitioning cellbeads and nucleic acid barcode molecules into partitions (e.g.,droplets, microwells) for analysis of cellular components (e.g., nucleicacid molecules) For example, in phase 1330, droplets comprising cellbeads from the pooled plurality of cell beads 1314 and beads (e.g., gelbeads) comprising nucleic acid barcode molecules 1322 (“barcode beads”)are generated such that at least some droplets comprise a cell bead anda barcode bead (e.g., a single cell bead and a single barcode bead). Asshown in FIG. 13D, an oil 1321, the cell beads 1314, and barcode beads1322 each comprising a barcode sequence (e.g., each bead comprising aunique barcode sequence) are provided to a microfluidic chip 1323. Anexemplary microfluidic chip architecture 1323 a is shown in FIG. 13C,but other suitable microfluidic chip architectures (e.g., FIGS. 4-7 )can also be utilized with the methods and systems disclosed herein. Asshown in FIG. 13C, the microfluidic chip 1323 comprises a plurality ofreservoirs comprising the oil 1321, cell beads 1314 and barcode beads1322 (e.g., gel beads). The chip also includes additional reservoirs1327 and 1328 that may be used to supply additional reagents (e.g.,reagents for nucleic acid amplification, reagents that can degrade ordissolve cell beads and/or gel beads, reagents that degrade linkagesbetween barcodes/cell bead index molecules and beads, reagents for celllysis, etc.). Cell beads 1314 and barcode beads 1322 are flowed (e.g.,via the action of an applied force, such as negative pressure via avacuum or positive pressure via a pump) from their reservoirs to a firstchannel junction and form an aqueous mixture. Materials from reservoirs1327 and 1328 can also be provided to the aqueous mixture at the firstchannel junction.

Alternatively, cell beads and barcode beads (e.g., gel beads) can bemixed before introduction into the microfluidic chip. In this case, asingle reservoir of the microfluidic chip (e.g., 1323) comprises amixture of cell beads and barcode beads. The ratio of cell beads tobarcode beads in the mixture can be varied to alter the number ofdroplets generated that comprise a single cell bead and a single barcodebead. The mixture of cell beads and barcode beads may be flowed (e.g.,via the action of an applied force, such as negative pressure via avacuum or positive pressure via a pump) from the reservoir to a firstchannel junction, in some cases together with materials from reservoirs1327 and/or 1328.

In some embodiments, the aqueous mixture comprising cell beads 1314,barcode beads 1321, and in some cases additional reagents is then flowedto a second channel junction, to which oil 1321 is provided. The aqueousmixture provided from the first channel junction is immiscible with theoil 1321 resulting in the generation of a suspension of aqueous droplets1325 in the oil which then flow to a reservoir for collection. Themicrofluidic chip can also include a reservoir 1329 that can acceptexcess oil from the stream emerging from the second channel. Flow can becontrolled within the microfluidic chip 1323 via any suitable strategy,including the use of one or more flow regulators in a channel or thatconnect channels, use of various channels, dimensioning of channels,etc. As shown in both FIG. 13A and FIG. 13C, the droplets 1325 comprisea cell bead 1314 and a barcode bead 1322 (e.g., a gel bead), in additionto any other reagents provided by reservoirs 1327 and 1328. In somecases, at least some droplets of droplets 1325 comprise a single cellbead and a single barcode bead (e.g., a single gel bead).

Where reagents that degrade or dissolve the cell beads 1314, barcodedbeads 1322 (e.g., gel beads) and/or linkages between barcodes andbarcoded beads 1322 are present in droplets, these reagents can releasethe nucleic acids trapped in the cell beads 1313, release the barcodesfrom the barcode beads 1322, and/or release cell bead index molecule(s)from the cell bead matrix. The nucleic acid barcode molecules caninteract with the released cellular components (e.g., cellular nucleicacids) and the cell bead index molecules to generate barcoded nucleicacid molecules for nucleic acid sequencing as described elsewhereherein. In embodiments where the barcode bead (e.g., gel bead) isdegraded or nucleic acid barcode molecules are releasably attached tothe barcode bead (e.g., gel bead), the barcoded cellular components(e.g., barcoded cDNA or gDNA fragments) are not attached to the bead.Where a given droplet comprises a cell bead (e.g., a single cell bead)and a barcoded bead (e.g., a single barcoded bead) comprising nucleicacid barcode molecules comprising a common barcode sequence, thebarcoded cellular components (or derivatives thereof) can be associatedwith the biological particle (e.g., a cell or other biological sample,such as a bacterium or virus) of the given cell bead via the commonbarcode sequence. Likewise, barcoded cell bead index moleculescomprising a common cell bead index sequence can be associated with agiven cell bead via the common barcode sequence while cell beadscomprising a common cell bead index sequence can be associated with agiven cell bead sample via the common cell bead index sequence.

Systems and Methods for Sample Compartmentalization

Disclosed herein, in some embodiments, are methods for cellularanalysis, comprising (a) providing a first cell bead comprising (i) afirst biological particle; and (ii) a nucleic acid molecule comprising afirst cell bead index sequence; (b) providing a second cell beadcomprising (i) a second biological particle; and (ii) a nucleic acidmolecule comprising a second cell bead index sequence different thansaid first cell bead index sequence; (c) pooling said first cell beadand said second cell bead to provide a pooled plurality of cell beads;and (d) partitioning said pooled plurality of cell beads into aplurality of partitions to generate (i) a first partition comprisingsaid first cell bead and a nucleic acid molecule comprising a firstbarcode sequence and (ii) a second partition comprising said second cellbead and a nucleic acid molecule comprising a second barcode sequence,wherein said second barcode sequence is different than said firstbarcode sequence. In some instances, the methods disclosed hereinfurther comprise: (e) generating (i) a first nucleic acid moleculecomprising said first cell bead index sequence and said first barcodesequence and (ii) a second nucleic acid molecule comprising said secondcell bead index sequence and said second barcode sequence.

Also disclosed herein, in some embodiments, are compositions comprising:(a) a first cell bead comprising (i) a first biological particle; and(ii) a nucleic acid molecule comprising a first cell bead indexsequence; and (b) a second cell bead comprising (i) a second biologicalparticle; and (ii) a nucleic acid molecule comprising a second cell beadindex sequence different than said first cell bead index sequence.

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, or partitioning of one or moreparticles (e.g., biological particles, macromolecular constituents ofbiological particles, cell beads, beads, reagents, etc.) into discretecompartments or partitions (referred to interchangeably herein aspartitions), where each partition maintains separation of its owncontents from the contents of other partitions. The partition can be adroplet in an emulsion. A partition may comprise one or more otherpartitions.

A partition may include one or more particles. A partition may includeone or more types of particles. For example, a partition of the presentdisclosure may comprise one or more biological particles and/ormacromolecular constituents thereof. A partition may comprise one ormore gel beads. A partition may comprise one or more cell beads. Apartition may include a single gel bead, a single cell bead, or both asingle cell bead and single gel bead. A partition may include one ormore reagents. Alternatively, a partition may be unoccupied. Forexample, a partition may not comprise a bead. A cell bead can be abiological particle and/or one or more of its macromolecularconstituents encased inside of a gel or polymer matrix, such as viapolymerization of a droplet containing the biological particle andprecursors capable of being polymerized or gelled. Unique identifiers,such as barcodes, may be partitioned into the partitions (e.g.,droplets) previous to, subsequent to, or concurrently with dropletgeneration, such as via a microcapsule (e.g., bead), as describedelsewhere herein. Microfluidic channel networks (e.g., on a chip) can beutilized to generate partitions as described herein. Alternativemechanisms may also be employed in the partitioning of individualbiological particles or cell beads, including porous membranes throughwhich aqueous mixtures of cells are extruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions may beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Patent Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual particlesto discrete partitions may in one non-limiting example be accomplishedby introducing a flowing stream of particles in an aqueous fluid into aflowing stream of a non-aqueous fluid, such that droplets are generatedat the junction of the two streams. Fluid properties (e.g., fluid flowrates, fluid viscosities, etc.), particle properties (e.g., volumefraction, particle size, particle concentration, etc.), microfluidicarchitectures (e.g., channel geometry, etc.), and other parameters maybe adjusted to control the occupancy of the resulting partitions (e.g.,number of biological particles or cell beads per partition, number ofbeads per partition, etc.). For example, partition occupancy can becontrolled by providing the aqueous stream at a certain concentrationand/or flow rate of particles. To generate single biological particlepartitions, the relative flow rates of the immiscible fluids can beselected such that, on average, the partitions may contain less than onebiological particle per partition in order to ensure that thosepartitions that are occupied are primarily singly occupied. In somecases, partitions among a plurality of partitions may contain at mostone biological particle (e.g., bead, DNA, cell or cellular material). Insome embodiments, the various parameters (e.g., fluid properties,particle properties, microfluidic architectures, etc.) may be selectedor adjusted such that a majority of partitions are occupied, forexample, allowing for only a small percentage of unoccupied partitions.The flows and channel architectures can be controlled as to ensure agiven number of singly occupied partitions, less than a certain level ofunoccupied partitions and/or less than a certain level of multiplyoccupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual biological particles or cell beads. In someinstances, the biological particles can be cells. The channel structure100 can include channel segments 102, 104, 106 and 108 communicating ata channel junction 110. In operation, a first aqueous fluid 112 thatincludes suspended biological particles (or cells) 114 may betransported along channel segment 102 into junction 110, while a secondfluid 116 that is immiscible with the aqueous fluid 112 is delivered tothe junction 110 from each of channel segments 104 and 106 to creatediscrete droplets 118, 120 of the first aqueous fluid 112 flowing intochannel segment 108, and flowing away from junction 110. The channelsegment 108 may be fluidically coupled to an outlet reservoir where thediscrete droplets can be stored and/or harvested. A discrete dropletgenerated may include an individual biological particle 114 (such asdroplets 118). A discrete droplet generated may include more than oneindividual biological particle 114 (not shown in FIG. 1 ). A discretedroplet may contain no biological particle 114 (such as droplet 120).Each discrete partition may maintain separation of its own contents(e.g., individual biological particle 114) from the contents of otherpartitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying particles (e.g., biological particles,cell beads, and/or gel beads) that meet at a channel junction. Fluid maybe directed to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114,and (2) unoccupied droplets 120, not containing any biological particles114. Occupied droplets 118 may comprise singly occupied droplets (havingone biological particle) and multiply occupied droplets (having morethan one biological particle). As described elsewhere herein, in somecases, the majority of occupied partitions can include no more than onebiological particle per occupied partition and some of the generatedpartitions can be unoccupied (of any biological particle). In somecases, though, some of the occupied partitions may include more than onebiological particle. In some cases, the partitioning process may becontrolled such that fewer than about 25% of the occupied partitionscontain more than one biological particle, and in many cases, fewer thanabout 20% of the occupied partitions have more than one biologicalparticle, while in some cases, fewer than about 10% or even fewer thanabout 5% of the occupied partitions include more than one biologicalparticle per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of biological particles (e.g., biological particles114) at the partitioning junction 110, such as to ensure that at leastone biological particle is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple biological particles. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the biological particles(e.g., in channel segment 102), or other fluids directed into thepartitioning junction (e.g., in channel segments 104, 106) can becontrolled such that, in many cases, no more than about 50% of thegenerated partitions, no more than about 25% of the generatedpartitions, or no more than about 10% of the generated partitions areunoccupied. These flows can be controlled so as to present anon-Poissonian distribution of single-occupied partitions whileproviding lower levels of unoccupied partitions. The above noted rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, in many cases,the use of the systems and methods described herein can create resultingpartitions that have multiple occupancy rates of less than about 25%,less than about 20%, less than about 15%, less than about 10%, and inmany cases, less than about 5%, while having unoccupied partitions ofless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles andadditional reagents, including, but not limited to, microcapsules orbeads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g.,oligonucleotides) (described in relation to FIG. 2 ). The occupiedpartitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% of the occupied partitions) can include both amicrocapsule (e.g., bead) comprising barcoded nucleic acid molecules anda biological particle.

Biological particles, such as cells or nuclei, may be encapsulatedwithin a microcapsule that comprises an outer shell, layer, or porousmatrix in which is entrained one or more individual biological particlesor small groups of biological particles (i.e., a cell bead). Themicrocapsule may include other reagents. Encapsulation of biologicalparticles may be performed by a variety of processes. Such processes maycombine an aqueous fluid containing the biological particles with apolymeric precursor material that may be capable of being formed into agel or other solid or semi-solid matrix upon application of a particularstimulus to the polymer precursor. Such stimuli can include, forexample, thermal stimuli (e.g., either heating or cooling),photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g.,through crosslinking, polymerization initiation of the precursor (e.g.,through added initiators)), mechanical stimuli, or a combinationthereof.

Preparation of microcapsules comprising biological particles (i.e., cellbeads) may be performed by a variety of methods. For example, air knifedroplet or aerosol generators may be used to dispense droplets ofprecursor fluids into gelling solutions in order to form microcapsulesthat include individual biological particles or small groups ofbiological particles. Likewise, membrane based encapsulation systems maybe used to generate microcapsules comprising encapsulated biologicalparticles as described herein. Microfluidic systems of the presentdisclosure, such as that shown in FIG. 1 , may be readily used inencapsulating cells as described herein. In particular, and withreference to FIG. 1 , the aqueous fluid 112 comprising (i) thebiological particles 114 and (ii) the polymer precursor material (notshown) is flowed into channel junction 110, where it is partitioned intodroplets 118, 120 through the flow of non-aqueous fluid 116. In the caseof encapsulation methods, non-aqueous fluid 116 may also include aninitiator (not shown) to cause polymerization and/or crosslinking of thepolymer precursor to form the microcapsule that includes the entrainedbiological particles. Examples of polymer precursor/initiator pairsinclude those described in U.S. Patent Application Publication No.2014/0378345, which is entirely incorporated herein by reference for allpurposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams 116 in channel segments 104 and 106, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the cells 114 (i.e. a cell bead). Althoughdescribed in terms of polyacrylamide encapsulation, other ‘activatable’encapsulation compositions may also be employed in the context of themethods and compositions described herein. For example, formation ofalginate droplets followed by exposure to divalent metal ions (e.g.,Ca²⁺ ions), can be used as an encapsulation process using the describedprocesses. Likewise, agarose droplets may also be transformed intocapsules through temperature based gelling (e.g., upon cooling, etc.).

In some cases, encapsulated biological particles (i.e. cell beads) canbe selectively releasable from the microcapsule, such as through passageof time or upon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the biological particles (e.g.,cell), or its other contents to be released from the microcapsule, suchas into a partition (e.g., droplet). For example, in the case of thepolyacrylamide polymer described above, degradation of the microcapsulemay be accomplished through the introduction of an appropriate reducingagent, such as DTT or the like, to cleave disulfide bonds thatcross-link the polymer matrix. See, for example, U.S. Patent ApplicationPublication No. 2014/0378345, which is entirely incorporated herein byreference for all purposes.

The biological particle can be subjected to other conditions sufficientto polymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, and/or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around thebiological particle. The polymer or gel may be diffusively permeable tochemical or biochemical reagents. The polymer or gel may be diffusivelyimpermeable to macromolecular constituents of the biological particle.In this manner, the polymer or gel may act to allow the biologicalparticle to be subjected to chemical or biochemical operations whilespatially confining the macromolecular constituents to a region of thedroplet defined by the polymer or gel. The polymer or gel may includeone or more of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel maycomprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. The polymer or gel may be stable in alkaline conditions or atelevated temperature. The polymer or gel may have mechanical propertiessimilar to the mechanical properties of the bead. For instance, thepolymer or gel may be of a similar size to the bead. The polymer or gelmay have a mechanical strength (e.g. tensile strength) similar to thatof the bead. The polymer or gel may be of a lower density than an oil.The polymer or gel may be of a density that is roughly similar to thatof a buffer. The polymer or gel may have a tunable pore size. The poresize may be chosen to, for instance, retain denatured nucleic acids. Thepore size may be chosen to maintain diffusive permeability to exogenouschemicals such as sodium hydroxide (NaOH) and/or endogenous chemicalssuch as inhibitors. The polymer or gel may be biocompatible. The polymeror gel may maintain or enhance cell viability. The polymer or gel may bebiochemically compatible. The polymer or gel may be polymerized and/ordepolymerized thermally, chemically, enzymatically, and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle may be surrounded by polyacrylamidestrands linked together by disulfide bridges. In this manner, thebiological particle may be encased inside of or comprise a gel or matrix(e.g., polymer matrix) to form a “cell bead.” A cell bead can containbiological particles (e.g., a cell) or macromolecular constituents(e.g., RNA, DNA, proteins, etc.) of biological particles. A cell beadmay include a single cell or multiple cells, or a derivative of thesingle cell or multiple cells. For example after lysing and washing thecells, inhibitory components from cell lysates can be washed away andthe macromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles.

Encapsulated biological particles can provide certain potentialadvantages of being more storable and more portable than droplet-basedpartitioned biological particles. Furthermore, in some cases, it may bedesirable to allow biological particles to incubate for a select periodof time before analysis, such as in order to characterize changes insuch biological particles over time, either in the presence or absenceof different stimuli. In such cases, encapsulation may allow for longerincubation than partitioning in emulsion droplets, although in somecases, droplet partitioned biological particles may also be incubatedfor different periods of time, e.g., at least 10 seconds, at least 30seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, atleast 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours,or at least 10 hours or more. The encapsulation of biological particlesmay constitute the partitioning of the biological particles into whichother reagents are co-partitioned. Alternatively or in addition,encapsulated biological particles may be readily deposited into otherpartitions (e.g., droplets) as described above.

Beads

A partition may comprise one or more unique identifiers, such asbarcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes may be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes maybe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can comprise a bead.Beads are described in further detail below.

In some cases, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus maydisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., with nucleic acid molecules, oligonucleotides, molecular tags)along the channel segment 201 into junction 210. The plurality of beads214 may be sourced from a suspension of beads. For example, the channelsegment 201 may be connected to a reservoir comprising an aqueoussuspension of beads 214. The channel segment 202 may transport theaqueous fluid 212 that includes a plurality of cell beads 216 along thechannel segment 202 into junction 210. The plurality of cell beads 216may be sourced from a suspension of cell beads. For example, the channelsegment 202 may be connected to a reservoir comprising an aqueoussuspension of cell beads 216. In some instances, the aqueous fluid 212in either the first channel segment 201 or the second channel segment202, or in both segments, can include one or more reagents, as furtherdescribed below. A second fluid 218 that is immiscible with the aqueousfluid 212 (e.g., oil) can be delivered to the junction 210 from each ofchannel segments 204 and 206. Upon meeting of the aqueous fluid 212 fromeach of channel segments 201 and 202 and the second fluid 218 from eachof channel segments 204 and 206 at the channel junction 210, the aqueousfluid 212 can be partitioned as discrete droplets 220 in the secondfluid 218 and flow away from the junction 210 along channel segment 208.The channel segment 208 may deliver the discrete droplets to an outletreservoir fluidly coupled to the channel segment 208, where they may beharvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads and cellbeads may form a mixture that is directed along another channel to thejunction 210 to yield droplets 220. The mixture may provide the beadsand cell beads in an alternating fashion, such that, for example, adroplet comprises a single bead and a single cell bead.

Beads, cell beads, and droplets may flow along channels at substantiallyregular flow profiles (e.g., at regular flow rates). Such regular flowprofiles may permit a droplet to include a single bead and a single cellbead. Such regular flow profiles may permit the droplets to have anoccupancy (e.g., droplets having beads and cell beads) greater than 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flowprofiles and devices that may be used to provide such regular flowprofiles are provided in, for example, U.S. Patent Publication No.2015/0292988, which is entirely incorporated herein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220.

A discrete droplet that is generated may include an individual cell bead216. A discrete droplet that is generated may include a barcode or otherreagent carrying bead 214. A discrete droplet generated may include bothan individual cell bead and a barcode carrying bead, such as droplets220. In some instances, a discrete droplet may include more than oneindividual cell bead or no cell bead. In some instances, a discretedroplet may include more than one bead or no bead. A discrete dropletmay be unoccupied (e.g., no beads, no cell beads).

Beneficially, a discrete droplet partitioning a cell bead and a barcodecarrying bead may effectively allow the attribution of the barcode tomacromolecular constituents of the cell bead within the partition. Thecontents of a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 10 nanometers (nm), 100 nm, 500nm, 1 micrometer (μall), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or greater. In somecases, a bead may have a diameter of less than about 10 nm, 100 nm, 500nm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm,90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or less. In some cases, a bead mayhave a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm,40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500μm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

A bead may comprise natural and/or synthetic materials. For example, abead (including the ell beads described herein) can comprise a naturalpolymer, a synthetic polymer or both natural and synthetic polymers.Examples of natural polymers include proteins and sugars such asdeoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose,amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the bead (including the cell beads described herein)may contain molecular precursors (e.g., monomers or polymers), which mayform a polymer network via polymerization of the molecular precursors.In some cases, a precursor may be an already polymerized species capableof undergoing further polymerization via, for example, a chemicalcross-linkage. In some cases, a precursor can comprise one or more of anacrylamide or a methacrylamide monomer, oligomer, or polymer. In somecases, the bead may comprise prepolymers, which are oligomers capable offurther polymerization. For example, polyurethane beads may be preparedusing prepolymers. In some cases, the bead may contain individualpolymers that may be further polymerized together. In some cases, beadsmay be generated via polymerization of different precursors, such thatthey comprise mixed polymers, co-polymers, and/or block co-polymers. Insome cases, the bead may comprise covalent or ionic bonds betweenpolymeric precursors (e.g., monomers, oligomers, linear polymers),nucleic acid molecules (e.g., oligonucleotides), primers, and otherentities. In some cases, the covalent bonds can be carbon-carbon bonds,thioether bonds, or carbon-heteroatom bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,cell bead index sequence, barcode sequence, barcoded nucleic acidmolecule, barcoded oligonucleotide, primer, or other oligonucleotide) tothe bead. In some cases, an acrydite moiety can refer to an acryditeanalogue generated from the reaction of acrydite with one or morespecies, such as, the reaction of acrydite with other monomers andcross-linkers during a polymerization reaction. Acrydite moieties may bemodified to form chemical bonds with a species to be attached, such as anucleic acid molecule (e.g., barcode sequence, barcoded nucleic acidmolecule, barcoded oligonucleotide, primer, or other oligonucleotide).Acrydite moieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides, barcodes, cell bead index molecules) may beachieved through a wide range of different approaches, includingactivation of chemical groups within a polymer, incorporation of activeor activatable functional groups in the polymer structure, or attachmentat the pre-polymer or monomer stage in bead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide, cell bead index molecule, barcode), which may include apriming sequence (e.g., a primer for amplifying target nucleic acids,random primer, primer sequence for messenger RNA) and/or one or morebarcode sequences. The one more barcode sequences may include sequencesthat are the same for all nucleic acid molecules coupled to a given beadand/or sequences that are different across all nucleic acid moleculescoupled to the given bead. The nucleic acid molecule may be incorporatedinto the bead.

In some cases, the nucleic acid molecule can comprise a functionalsequence, for example, for attachment to a sequencing flow cell, suchas, for example, a P5 sequence for Illumina® sequencing. In some cases,the nucleic acid molecule or derivative thereof (e.g., oligonucleotideor polynucleotide generated from the nucleic acid molecule) can compriseanother functional sequence, such as, for example, a P7 sequence forattachment to a sequencing flow cell for Illumina sequencing. In somecases, the nucleic acid molecule can comprise a barcode sequence. Insome cases, the primer can further comprise a unique molecularidentifier (UMI). In some cases, the primer can comprise an R1 primersequence for Illumina sequencing. In some cases, the primer can comprisean R2 primer sequence for Illumina sequencing. Examples of such nucleicacid molecules (e.g., oligonucleotides, polynucleotides, etc.) and usesthereof, as may be used with compositions, devices, methods and systemsof the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporatedherein by reference.

FIG. 8 illustrates an example of a barcode carrying bead (or of a cellbead comprising cell bead index molecules). A nucleic acid molecule 802,such as an oligonucleotide, can be coupled to a bead 804 by an optionalreleasable linkage 806, such as, for example, a disulfide linker. Insome embodiments, the nucleic acid molecule 802, can be coupled to abead through a covalent, non-releasable linkage. The same bead 804 maybe coupled (e.g., via releasable or non-releasable linkage) to one ormore other nucleic acid molecules 818, 820. The nucleic acid molecule802 may be or comprise a barcode sequence. As noted elsewhere herein,the structure of the barcode may comprise a number of sequence elements.The nucleic acid molecule 802 may comprise a functional sequence 808that may be used in subsequent processing. For example, the functionalsequence 808 may include one or more of a sequencer specific flow cellattachment sequence (e.g., a P5 sequence for Illumina® sequencingsystems) and a sequencing primer sequence (e.g., a R1 primer forIllumina® sequencing systems). The nucleic acid molecule 802 maycomprise a barcode sequence 810 for use in barcoding the sample (e.g.,DNA, RNA, protein, cell bead index molecule, etc.). In some cases, thebarcode sequence 810 can be bead-specific such that the barcode sequence810 is common to all nucleic acid molecules (e.g., including nucleicacid molecule 802) coupled to the same bead 804. Alternatively, or inaddition, the barcode sequence 810 can be partition-specific such thatthe barcode sequence 810 is common to all nucleic acid molecules coupledto one or more beads that are partitioned into the same partition. Thenucleic acid molecule 802 may comprise a specific priming sequence 812,such as an mRNA specific priming sequence (e.g., poly-T sequence), atargeted priming sequence, and/or a random priming sequence. The nucleicacid molecule 802 may comprise an anchoring sequence 814 to ensure thatthe specific priming sequence 812 hybridizes at the sequence end (e.g.,of the mRNA). For example, the anchoring sequence 814 can include arandom short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer orlonger sequence, which can ensure that a poly-T segment is more likelyto hybridize at the sequence end of the poly-A tail of the mRNA.

The nucleic acid molecule 802 may comprise a unique molecularidentifying sequence 816 (e.g., unique molecular identifier (UMI)). Insome cases, the unique molecular identifying sequence 816 may comprisefrom about 5 to about 8 nucleotides. Alternatively, the unique molecularidentifying sequence 816 may compress less than about 5 or more thanabout 8 nucleotides. The unique molecular identifying sequence 816 maybe a unique sequence that varies across individual nucleic acidmolecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g.,bead 804). In some cases, the unique molecular identifying sequence 816may be a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI may provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantitation of the numberof original expressed RNA. As will be appreciated, although FIG. 8 showsthree nucleic acid molecules 802, 818, 820 coupled to the surface of thebead 804, an individual bead may be coupled to any number of individualnucleic acid molecules, for example, from one to tens to hundreds ofthousands or even millions of individual nucleic acid molecules. Therespective barcodes for the individual nucleic acid molecules cancomprise both common sequence segments or relatively common sequencesegments (e.g., 808, 810, 812, etc.) and variable or unique sequencesegments (e.g., 816) between different individual nucleic acid moleculescoupled to the same bead.

In operation, a cell bead can be co-partitioned along with a barcodebearing bead 804. In some embodiment, the barcoded nucleic acidmolecules 802, 818, 820 and/or cell bead index molecules can be releasedfrom the bead 804 in the partition. By way of example, in the context ofanalyzing sample RNA, the poly-T segment (e.g., 812) of one of thereleased nucleic acid molecules (e.g., 802) can hybridize to the poly-Atail of a mRNA molecule. Reverse transcription may result in a cDNAtranscript of the mRNA, but which transcript includes each of thesequence segments 808, 810, 816 of the nucleic acid molecule 802.Because the nucleic acid molecule 802 comprises an anchoring sequence814, it will more likely hybridize to and prime reverse transcription atthe sequence end of the poly-A tail of the mRNA. Within any givenpartition, all of the cDNA transcripts of the individual mRNA molecules(as well as the cell bead index molecules) may include a common barcodesequence segment 810. However, the transcripts made from the differentmRNA molecules within a given partition may vary at the unique molecularidentifying sequence 812 segment (e.g., UMI segment). Beneficially, evenfollowing any subsequent amplification of the contents of a givenpartition, the number of different UMIs can be indicative of thequantity of mRNA originating from a given partition, and thus from thebiological particle (e.g., cell). As noted above, the transcripts can beamplified, cleaned up and sequenced to identify the sequence of the cDNAtranscript of the mRNA, as well as to sequence the barcode segment andthe UMI segment. While a poly-T primer sequence is described, othertargeted or random priming sequences may also be used in priming thereverse transcription reaction. Likewise, although described asreleasing the barcoded oligonucleotides into the partition, in somecases, the nucleic acid molecules bound to the bead (e.g., gel bead) maybe used to hybridize and capture the mRNA on the solid phase of thebead, for example, in order to facilitate the separation of the RNA fromother cell contents.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads or cell beadscomprising the activated or activatable functional group. The functionalgroup may then be used to attach additional species (e.g., disulfidelinkers, primers, other oligonucleotides, cell bead index molecules,etc.) to the gel or cell beads. For example, some precursors comprisinga carboxylic acid (COOH) group can co-polymerize with other precursorsto form a gel or cell bead that also comprises a COOH functional group.In some cases, acrylic acid (a species comprising free COOH groups),acrylamide, and bis(acryloyl)cystamine can be co-polymerized together togenerate a gel or cell bead comprising free COOH groups. The COOH groupsof the gel or cell bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species (e.g., cell bead indexmolecules) comprising the acrydite is linked to the bead (e.g., cellbead). In some cases, uncontrolled reactions can be prevented byinclusion of a thiol capping agent such as N-ethylmalieamide oriodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead pr cell bead after gelbead or cell bead formation may be advantageous. For example, additionof an oligonucleotide (e.g., barcoded oligonucleotide) after gel or cellbead formation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel or cell bead synthesis canminimize exposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide (e.g.,barcodes or cell bead index molecules). Post-productionfunctionalization may also be useful in controlling loading ratios ofspecies in beads, such that, for example, the variability in loadingratio is minimized. Species loading may also be performed in a batchprocess such that a plurality of beads can be functionalized with thespecies in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes or cell beadindex molecules. A bead injected or otherwise introduced into apartition may comprise activatable barcodes. A bead injected orotherwise introduced into a partition may be degradable, disruptable, ordissolvable beads.

Barcodes or cell bead index molecules can be releasably, cleavably orreversibly attached to the beads such that barcodes can be released orbe releasable through cleavage of a linkage between the barcode moleculeand the bead, or released through degradation of the underlying beaditself, allowing the barcodes to be accessed or be accessible by otherreagents, or both. In non-limiting examples, cleavage may be achievedthrough reduction of di-sulfide bonds, use of restriction enzymes,photo-activated cleavage, or cleavage via other types of stimuli (e.g.,chemical, thermal, pH, enzymatic, etc.) and/or reactions, such asdescribed elsewhere herein. Releasable barcodes (or cell bead indexmolecules) may sometimes be referred to as being activatable, in thatthey are available for reaction once released. Thus, for example, anactivatable barcode may be activated by releasing the barcode from abead (or other suitable type of partition described herein). Otheractivatable configurations are also envisioned in the context of thedescribed methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead or cell beadcan be degraded or dissolved at elevated temperature and/or in basicconditions. In some cases, a bead may be thermally degradable such thatwhen the bead is exposed to an appropriate change in temperature (e.g.,heat), the bead degrades. Degradation or dissolution of a bead bound toa species (e.g., a nucleic acid molecule, e.g., barcoded oligonucleotideor cellular constituents of a cell bead) may result in release of thespecies from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the disassociation of a bound or entrained speciesfrom a bead, both with and without structurally degrading the physicalbead itself. For example, the degradation of the bead may involvecleavage of a cleavable linkage via one or more species and/or methodsdescribed elsewhere herein. In another example, entrained species may bereleased from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself. In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the partition (e.g., droplet) when the appropriatestimulus is applied. The free species (e.g., oligonucleotides, nucleicacid molecules) may interact with other reagents contained in thepartition. For example, a polyacrylamide bead comprising cystamine andlinked, via a disulfide bond, to a barcode sequence, may be combinedwith a reducing agent within a droplet of a water-in-oil emulsion.Within the droplet, the reducing agent can break the various disulfidebonds, resulting in bead degradation and release of the barcode sequenceinto the aqueous, inner environment of the droplet. In another example,heating of a droplet comprising a bead-bound barcode sequence in basicsolution may also result in bead degradation and release of the attachedbarcode sequence into the aqueous, inner environment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingnucleic acid molecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photo-sensitive bond e.g., disulfidebond, UV sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, cell bead index molecules, etc.) to a bead. In somecases, a thermally labile bond may include a nucleic acidhybridization-based attachment, e.g., where an oligonucleotide ishybridized to a complementary sequence that is attached to the bead,such that thermal melting of the hybrid releases the oligonucleotide,e.g., a barcode containing sequence, from the bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead and/or cellbead may result in the generation of a bead capable of responding tovaried stimuli. Each type of labile bond may be sensitive to anassociated stimulus (e.g., chemical stimulus, light, temperature,enzymatic, etc.) such that release of species attached to a bead viaeach labile bond may be controlled by the application of the appropriatestimulus. Such functionality may be useful in controlled release ofspecies from a gel bead and/or cell bead. In some cases, another speciescomprising a labile bond may be linked to a gel or cell bead after gelbead formation via, for example, an activated functional group of thegel bead as described above. As will be appreciated, barcodes that arereleasably, cleavably or reversibly attached to the beads describedherein include barcodes that are released or releasable through cleavageof a linkage between the barcode molecule and the bead, or that arereleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or accessible by other reagents, or both.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel or cell beads after formation. Such species may include, forexample, nucleic acid molecules (e.g., oligonucleotides), reagents for anucleic acid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., Nextera® for Illumina®). Such species may include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNAse, etc. Suchspecies may include one or more reagents described elsewhere herein(e.g., lysis agents, inhibitors, inactivating agents, chelating agents,stimulus). Trapping of such species may be controlled by the polymernetwork density generated during polymerization of precursors, controlof ionic charge within the gel or cell bead (e.g., via ionic specieslinked to polymerized species), or by the release of other species.Encapsulated species may be released from a bead upon bead degradationand/or by application of a stimulus capable of releasing the speciesfrom the bead. Alternatively or in addition, species may be partitionedin a partition (e.g., droplet) during or subsequent to partitionformation. Such species may include, without limitation, theabovementioned species that may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attached orentrapped species (e.g., a nucleic acid molecule, a barcode sequence, aprimer, etc) from the bead when the appropriate stimulus is applied tothe bead as compared to a bead that does not degrade. For example, for aspecies bound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence (e.g., cell bead index) may be attached, via a disulfide bond,to a polyacrylamide bead comprising cystamine. Upon exposure of thebarcoded-bead to a reducing agent, the bead degrades and the barcodesequence is released upon breakage of both the disulfide linkage betweenthe barcode sequence and the bead and the disulfide linkages of thecystamine in the bead.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead. In some cases, any combination of stimulimay trigger degradation of a bead. For example, a change in pH mayenable a chemical agent (e.g., DTT) to become an effective reducingagent.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingoligonucleotide bearing beads.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the biological particle and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a given occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of cell beads (e.g., one cell bead and one bead per partition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL,20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedcell beads and/or beads, within the partitions may be less than about90% of the above described volumes, less than about 80%, less than about70%, less than about 60%, less than about 50%, less than about 40%, lessthan about 30%, less than about 20%, or less than about 10% of the abovedescribed volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000,000 partitions, at least about5,000,000 partitions at least about 10,000,000 partitions, at leastabout 50,000,000 partitions, at least about 100,000,000 partitions, atleast about 500,000,000 partitions, at least about 1,000,000,000partitions, or more partitions can be generated or otherwise provided.Moreover, the plurality of partitions may comprise both unoccupiedpartitions (e.g., empty partitions) and occupied partitions.

Reagents

In accordance with certain aspects, biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to, the introduction of thebiological particles into the partitioning junction/droplet generationzone (e.g., junction 210), such as through an additional channel orchannels upstream of the channel junction. In accordance with otheraspects, additionally or alternatively, biological particles may bepartitioned along with other reagents, as will be described furtherbelow.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning biological particles and reagents (e.g., to generate acell bead). The channel structure 300 can include channel segments 301,302, 304, 306 and 308. Channel segments 301 and 302 communicate at afirst channel junction 309. Channel segments 302, 304, 306, and 308communicate at a second channel junction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of biological particles 314along the channel segment 301 into the second junction 310. As analternative or in addition to, channel segment 301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of biological particles 314. Upstreamof, and immediately prior to reaching, the second junction 310, thechannel segment 301 may meet the channel segment 302 at the firstjunction 309. The channel segment 302 may transport a plurality ofreagents 315 (e.g., lysis agents) suspended in the aqueous fluid 312along the channel segment 302 into the first junction 309. For example,the channel segment 302 may be connected to a reservoir comprising thereagents 315. After the first junction 309, the aqueous fluid 312 in thechannel segment 301 can carry both the biological particles 314 and thereagents 315 towards the second junction 310. In some instances, theaqueous fluid 312 in the channel segment 301 can include one or morereagents, which can be the same or different reagents as the reagents315. A second fluid 316 that is immiscible with the aqueous fluid 312(e.g., oil) can be delivered to the second junction 310 from each ofchannel segments 304 and 306. Upon meeting of the aqueous fluid 312 fromthe channel segment 301 and the second fluid 316 from each of channelsegments 304 and 306 at the second channel junction 310, the aqueousfluid 312 can be partitioned as discrete droplets 318 in the secondfluid 316 and flow away from the second junction 310 along channelsegment 308. The channel segment 308 may deliver the discrete droplets318 to an outlet reservoir fluidly coupled to the channel segment 308,where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual biologicalparticle 314 and/or one or more reagents 315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles areco-partitioned, the lysis reagents can facilitate the release of thecontents of the biological particles within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or biological particles that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, MO), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the biological particles to causethe release of the biological particles's contents into the partitions.For example, in some cases, surfactant-based lysis solutions may be usedto lyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanicalcellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of biologicalparticles that may be in addition to or in place of dropletpartitioning, where any pore size of the encapsulate is sufficientlysmall to retain nucleic acid fragments of a given size, followingcellular disruption.

Alternatively or in addition to the lysis agents co-partitioned with thebiological particles described above, other reagents can also beco-partitioned with the biological particles, including, for example,DNase and RNase inactivating agents or inhibitors, such as proteinase K,chelating agents, such as EDTA, and other reagents employed in removingor otherwise reducing negative activity or impact of different celllysate components on subsequent processing of nucleic acids. Inaddition, in the case of encapsulated biological particles, thebiological particles may be exposed to an appropriate stimulus torelease the biological particles or their contents from a co-partitionedmicrocapsule. For example, in some cases, a chemical stimulus may beco-partitioned along with an encapsulated biological particle to allowfor the degradation of the microcapsule and release of the cell or itscontents into the larger partition. In some cases, this stimulus may bethe same as the stimulus described elsewhere herein for release ofnucleic acid molecules (e.g., oligonucleotides) from their respectivemicrocapsule (e.g., bead). In alternative aspects, this may be adifferent and non-overlapping stimulus, in order to allow anencapsulated biological particle to be released into a partition at adifferent time from the release of nucleic acid molecules into the samepartition.

Additional reagents may also be co-partitioned with the biologicalparticles, such as endonucleases to fragment a biological particle'sDNA, DNA polymerase enzymes and dNTPs used to amplify the biologicalparticle's nucleic acid fragments and to attach the barcode moleculartags to the amplified fragments. Other enzymes may be co-partitioned,including without limitation, polymerase, transposase, ligase,proteinase K, DNAse, etc. Additional reagents may also include reversetranscriptase enzymes, including enzymes with terminal transferaseactivity, primers and oligonucleotides, and switch oligonucleotides(also referred to herein as “switch oligos” or “template switchingoligonucleotides”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In some cases, template switching can be used to append a predefinednucleic acid sequence to the cDNA. In an example of template switching,cDNA can be generated from reverse transcription of a template, e.g.,cellular mRNA, where a reverse transcriptase with terminal transferaseactivity can add additional nucleotides, e.g., polyC, to the cDNA in atemplate independent manner. Switch oligos can include sequencescomplementary to the additional nucleotides, e.g., polyG. The additionalnucleotides (e.g., polyC) on the cDNA can hybridize to the additionalnucleotides (e.g., polyG) on the switch oligo, whereby the switch oligocan be used by the reverse transcriptase as template to further extendthe cDNA. Template switching oligonucleotides may comprise ahybridization region and a template region. The hybridization region cancomprise any sequence capable of hybridizing to the target. In somecases, as previously described, the hybridization region comprises aseries of G bases to complement the overhanging C bases at the 3′ end ofa cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases,3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxyInosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual biological particles canbe provided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same biological particle or particles. The ability toattribute characteristics to individual biological particles or groupsof biological particles is provided by the assignment of uniqueidentifiers specifically to an individual biological particle or groupsof biological particles. Unique identifiers, e.g., in the form ofnucleic acid barcodes can be assigned or associated with individualbiological particles or populations of biological particles, in order totag or label the biological particle's macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle'scomponents and characteristics to an individual biological particle orgroup of biological particles.

In some aspects, this is performed by co-partitioning the individualbiological particle or groups of biological particles with the uniqueidentifiers, such as described above (with reference to FIG. 2 ). Insome aspects, the unique identifiers are provided in the form of nucleicacid molecules (e.g., oligonucleotides) that comprise nucleic acidbarcode sequences that may be attached to or otherwise associated withthe nucleic acid contents of individual biological particle, or to othercomponents of the biological particle, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). The nucleic acid barcode sequences can includefrom about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides. In some cases, the length of a barcode sequence may beabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotidesor longer. In some cases, the length of a barcode sequence may be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some cases, the length of a barcode sequencemay be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 nucleotides or shorter. These nucleotides may be completelycontiguous, i.e., in a single stretch of adjacent nucleotides, or theymay be separated into two or more separate subsequences that areseparated by 1 or more nucleotides. In some cases, separated barcodesubsequences can be from about 4 to about 16 nucleotides in length. Insome cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some cases, the barcode subsequence maybe at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned biological particles. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual biological particleswithin the partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down barcoded nucleic acids, or any of a number of otherpotential functional sequences. Other mechanisms of co-partitioningoligonucleotides may also be employed, including, e.g., coalescence oftwo or more droplets, where one droplet contains oligonucleotides, ormicrodispensing of oligonucleotides into partitions, e.g., dropletswithin microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) releasably attached to thebeads, where all of the nucleic acid molecules attached to a particularbead will include the same nucleic acid barcode sequence, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. In some embodiments, hydrogel beads, e.g.,comprising polyacrylamide polymer matrices, are used as a solid supportand delivery vehicle for the nucleic acid molecules into the partitions,as they are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules uponexposure to a particular stimulus, as described elsewhere herein. Insome cases, the population of beads provides a diverse barcode sequencelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of nucleic acid (e.g., oligonucleotide) moleculesattached. In particular, the number of molecules of nucleic acidmolecules including the barcode sequence on an individual bead can be atleast about 1,000 nucleic acid molecules, at least about 5,000 nucleicacid molecules, at least about 10,000 nucleic acid molecules, at leastabout 50,000 nucleic acid molecules, at least about 100,000 nucleic acidmolecules, at least about 500,000 nucleic acids, at least about1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acidmolecules, at least about 10,000,000 nucleic acid molecules, at leastabout 50,000,000 nucleic acid molecules, at least about 100,000,000nucleic acid molecules, at least about 250,000,000 nucleic acidmolecules and in some cases at least about 1 billion nucleic acidmolecules, or more. Nucleic acid molecules of a given bead can includeidentical (or common) barcode sequences, different barcode sequences, ora combination of both. Nucleic acid molecules of a given bead caninclude multiple sets of nucleic acid molecules. Nucleic acid moleculesof a given set can include identical barcode sequences. The identicalbarcode sequences can be different from barcode sequences of nucleicacid molecules of another set.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000,000 nucleic acid molecules, at least about 5,000,000nucleic acid molecules, at least about 10,000,000 nucleic acidmolecules, at least about 50,000,000 nucleic acid molecules, at leastabout 100,000,000 nucleic acid molecules, at least about 250,000,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) are releasable fromthe beads upon the application of a particular stimulus to the beads. Insome cases, the stimulus may be a photo-stimulus, e.g., through cleavageof a photo-labile linkage that releases the nucleic acid molecules. Inother cases, a thermal stimulus may be used, where elevation of thetemperature of the beads environment will result in cleavage of alinkage or other release of the nucleic acid molecules form the beads.In still other cases, a chemical stimulus can be used that cleaves alinkage of the nucleic acid molecules to the beads, or otherwise resultsin release of the nucleic acid molecules from the beads. In one case,such compositions include the polyacrylamide matrices described abovefor encapsulation of biological particles, and may be degraded forrelease of the attached nucleic acid molecules through exposure to areducing agent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the junction 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the junction 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, α, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the junction 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or morebiological particles, as described elsewhere herein. In some instances,a discrete droplet generated may comprise one or more reagents, asdescribed elsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4 ). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise biological particles or cell beads (e.g., described withreference to FIGS. 1 and 2 ). In some instances, the aqueous fluid 408can have a substantially uniform concentration or frequency ofbiological particles or cell beads. As with the beads, the biologicalparticles or cell beads can be introduced into the channel segment 402from a separate channel. The frequency or concentration of thebiological particles in the aqueous fluid 408 in the channel segment 402may be controlled by controlling the frequency in which the biologicalparticles are introduced into the channel segment 402 and/or therelative flow rates of the fluids in the channel segment 402 and theseparate channel. In some instances, the biological particles can beintroduced into the channel segment 402 from a plurality of differentchannels, and the frequency controlled accordingly. In some instances, afirst separate channel can introduce beads and a second separate channelcan introduce biological particles into the channel segment 402. Thefirst separate channel introducing the beads may be upstream ordownstream of the second separate channel introducing the biologicalparticles.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the junction 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the junction 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, h₀ and width, w, at or near the junction 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the junction 406 can beinclined at an expansion angle, α. The expansion angle, α, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and α:

$R_{d} \approx {{0.4}4( {1 + {{2.2}\sqrt{\tan\alpha}\frac{w}{h_{0}}}} )\frac{h_{0}}{\sqrt{\tan\alpha}}}$

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 μm, h=25 μm, and α=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, α, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°,55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.In some instances, the width, w, can be between a range of from about100 micrometers (μm) to about 500 μm. In some instances, the width, w,can be between a range of from about 10 μm to about 200 μm.Alternatively, the width can be less than about 10 μm. Alternatively,the width can be greater than about 500 μm. In some instances, the flowrate of the aqueous fluid 408 entering the junction 406 can be betweenabout 0.04 microliters (μL)/minute (min) and about 40 μL/min. In someinstances, the flow rate of the aqueous fluid 408 entering the junction406 can be between about 0.01 microliters (μL)/minute (min) and about100 μL/min. Alternatively, the flow rate of the aqueous fluid 408entering the junction 406 can be less than about 0.01 μL/min.Alternatively, the flow rate of the aqueous fluid 408 entering thejunction 406 can be greater than about 40 μL/min, such as 45 μL/min, 50μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110 μL/min, 120μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. At lower flowrates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 408 entering the junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctions. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejunction where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe junction, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctions 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 2 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctions. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the junction where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the junction, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctions 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, a (not shown in FIG. 6 )at or near each channel junction. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6 ) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/orbiological particle injected into the droplets may or may not haveuniform size.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 700 can include a channel segment 702 communicatingat a channel junction 706 (or intersection) with a reservoir 704. Insome instances, the channel structure 700 and one or more of itscomponents can correspond to the channel structure 100 and one or moreof its components. FIG. 7B shows a perspective view of the channelstructure 700 of FIG. 7A.

An aqueous fluid 712 comprising a plurality of particles 716 may betransported along the channel segment 702 into the junction 706 to meeta second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueousfluid 712 in the reservoir 704 to create droplets 720 of the aqueousfluid 712 flowing into the reservoir 704. At the junction 706 where theaqueous fluid 712 and the second fluid 714 meet, droplets can form basedon factors such as the hydrodynamic forces at the junction 706, relativeflow rates of the two fluids 712, 714, fluid properties, and certaingeometric parameters (e.g., Δh, etc.) of the channel structure 700. Aplurality of droplets can be collected in the reservoir 704 bycontinuously injecting the aqueous fluid 712 from the channel segment702 at the junction 706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 712 can have a substantiallyuniform concentration or frequency of particles 716. As describedelsewhere herein (e.g., with reference to FIG. 4 ), the particles 716(e.g., beads) can be introduced into the channel segment 702 from aseparate channel (not shown in FIG. 7 ). The frequency of particles 716in the channel segment 702 may be controlled by controlling thefrequency in which the particles 716 are introduced into the channelsegment 702 and/or the relative flow rates of the fluids in the channelsegment 702 and the separate channel. In some instances, the particles716 can be introduced into the channel segment 702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce biological particles into the channelsegment 702. The first separate channel introducing the beads may beupstream or downstream of the second separate channel introducing thebiological particles.

In some instances, the second fluid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond fluid 714 may be substantially stationary in the reservoir 704.In some instances, the second fluid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous fluid 712 at the junction 706.Alternatively, the second fluid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second fluid 714 from upstream to downstream,transporting the generated droplets.

The channel structure 700 at or near the junction 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 700. The channelsegment 702 can have a first cross-section height, h₁, and the reservoir704 can have a second cross-section height, h₂. The first cross-sectionheight, h₁, and the second cross-section height, h₂, may be different,such that at the junction 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. In some instances, the reservoir maythereafter gradually increase in cross-section height, for example, themore distant it is from the junction 706. In some instances, thecross-section height of the reservoir may increase in accordance withexpansion angle, β, at or near the junction 706. The height difference,Δh, and/or expansion angle, β, can allow the tongue (portion of theaqueous fluid 712 leaving channel segment 702 at junction 706 andentering the reservoir 704 before droplet formation) to increase indepth and facilitate decrease in curvature of the intermediately formeddroplet. For example, droplet size may decrease with increasing heightdifference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 μm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 712 entering thejunction 706 can be between about 0.04 microliters (μL)/minute (min) andabout 40 μt/min. In some instances, the flow rate of the aqueous fluid712 entering the junction 706 can be between about 0.01 microliters(μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate ofthe aqueous fluid 712 entering the junction 706 can be less than about0.01 μL/min. Alternatively, the flow rate of the aqueous fluid 712entering the junction 706 can be greater than about 40 μL/min, such as45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. Atlower flow rates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 712 entering the junction 706. The secondfluid 714 may be stationary, or substantially stationary, in thereservoir 704. Alternatively, the second fluid 714 may be flowing, suchas at the above flow rates described for the aqueous fluid 712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the junction 706 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 706, a bottom wall of thechannel and a bottom wall of the reservoir can meet at an angle greaterthan 90°. Alternatively or in addition, a top wall (e.g., ceiling) ofthe channel and a top wall (e.g., ceiling) of the reservoir can meet anangle greater than 90°. A gradual increase or decrease may be linear ornon-linear (e.g., exponential, sinusoidal, etc.). Alternatively or inaddition, the height difference may variably increase and/or decreaselinearly or non-linearly. While FIGS. 7A and 7B illustrate the expandingreservoir cross-section height as linear (e.g., constant expansionangle, β), the cross-section height may expand non-linearly. Forexample, the reservoir may be defined at least partially by a dome-like(e.g., hemispherical) shape having variable expansion angles. Thecross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 208,reservoir 604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 9 shows a computer system 901that is programmed or otherwise configured to implement methods or partsof methods described herein, such as analysis of multiplexed samplescomprising two or more cell types. The computer system 901 can regulatevarious aspects of the present disclosure, such as, for example, samplepreparation of cellular materials in cell beads, barcoding of thesematerials and/or analysis of barcoded molecules. The computer system 901can be an electronic device of a user or a computer system that isremotely located with respect to the electronic device. The electronicdevice can be a mobile electronic device.

The computer system 901 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 905, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 901 also includes memory or memorylocation 910 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 915 (e.g., hard disk), communicationinterface 920 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 925, such as cache, other memory,data storage and/or electronic display adapters. The memory 910, storageunit 915, interface 920 and peripheral devices 925 are in communicationwith the CPU 905 through a communication bus (solid lines), such as amotherboard. The storage unit 915 can be a data storage unit (or datarepository) for storing data. The computer system 901 can be operativelycoupled to a computer network (“network”) 930 with the aid of thecommunication interface 920. The network 930 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 930 in some cases is atelecommunication and/or data network. The network 930 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. The network 930, in some cases with the aid of thecomputer system 901, can implement a peer-to-peer network, which mayenable devices coupled to the computer system 901 to behave as a clientor a server.

The CPU 905 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 910. The instructionscan be directed to the CPU 905, which can subsequently program orotherwise configure the CPU 905 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 905 can includefetch, decode, execute, and writeback.

The CPU 905 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 901 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 915 can store files, such as drivers, libraries andsaved programs. The storage unit 915 can store user data, e.g., userpreferences and user programs. The computer system 901 in some cases caninclude one or more additional data storage units that are external tothe computer system 901, such as located on a remote server that is incommunication with the computer system 901 through an intranet or theInternet.

The computer system 901 can communicate with one or more remote computersystems through the network 930. For instance, the computer system 901can communicate with a remote computer system of a user (e.g.,operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 901 via the network 930.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 901, such as, for example, on the memory910 or electronic storage unit 915. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 905. In some cases, the code canbe retrieved from the storage unit 915 and stored on the memory 910 forready access by the processor 905. In some situations, the electronicstorage unit 915 can be precluded, and machine-executable instructionsare stored on memory 910.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 901, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 901 can include or be in communication with anelectronic display 935 that comprises a user interface (UI) 940 forproviding, for example, results of sequencing analysis of nucleic acidsand their associated cell type from a multiplexed sampled comprising twoor more cell types. Examples of UIs include, without limitation, agraphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 905. Thealgorithm can, for example, perform sequencing analysis of nucleic acidsand their associated cell type from a multiplexed sampled comprising twoor more cell types.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)from a single cell. For example, a biological particle (e.g., a cell orcell bead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the biological particle are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for cellular analysis, comprising: (a)providing a first cell bead comprising: (i) a first cell or a first cellnucleus contained within in a first hydrogel, a first polymericmaterial, or a first cross-linked material; and (ii) a first cell beadindex nucleic acid molecule attached to the first hydrogel, the firstpolymeric material, or the first cross-linked material, wherein thefirst cell bead index nucleic acid molecule comprises a first cell beadindex sequence; (b) providing a second cell bead comprising: (i) asecond cell or a second cell nucleus contained within in a secondhydrogel, a second polymeric material, or a second cross-linkedmaterial; and (ii) a second cell bead index nucleic acid moleculeattached to the second hydrogel, the second polymeric material, or thesecond cross-linked material, wherein the second cell bead index nucleicacid molecule comprises a second cell bead index sequence different fromthe first cell bead index sequence; (c) producing a pooled plurality ofcell beads comprising the first cell bead and the second cell bead bypooling the first cell bead and the second cell bead together; and (d)co-partitioning cell beads of the pooled plurality of cell beads andbeads comprising nucleic acid barcode molecules into a plurality ofpartitions, wherein the plurality of partitions comprises: (i) a firstpartition comprising the first cell bead and a first bead comprising afirst nucleic acid barcode molecule from the beads comprising nucleicacid barcode molecules, wherein the first nucleic acid barcode moleculecomprises a first barcode sequence, and (ii) a second partitioncomprising the second cell bead and a second bead comprising a secondnucleic acid barcode molecule from the beads comprising nucleic acidbarcode molecules, wherein the second nucleic acid barcode moleculecomprises a second barcode sequence, and wherein said second barcodesequence is different from the first barcode sequence.
 2. The method ofclaim 1, further comprising (e) generating a first nucleic acid moleculecomprising the first cell bead index sequence and the first barcodesequence using the first cell bead index nucleic acid molecule and thefirst nucleic acid barcode molecule, and generating a second nucleicacid molecule comprising the second cell bead index sequence and thesecond barcode sequence using the second cell bead index nucleic acidmolecule and the second nucleic acid barcode molecule.
 3. The method ofclaim 1, wherein the first cell bead index sequence identifies the firstcell bead and wherein the second cell bead index sequence identifies thesecond cell bead.
 4. The method of claim 1, further comprisinggenerating a first plurality of cell beads including the first cell beadbefore (a), and wherein each cell bead of the first plurality of cellbeads comprises a cell or a cell nucleus and the first cell bead indexsequence, and generating a second plurality of cell beads including thesecond cell bead before (b), wherein each cell bead of the secondplurality of cell beads comprises a cell or a cell nucleus and thesecond cell bead index sequence.
 5. The method of claim 4, wherein saidpooling the first cell bead and the second cell bead is performed bypooling the first plurality of cell beads and the second plurality ofcell beads.
 6. The method of claim 1, further comprising (e) generating(i) a first barcoded molecule comprising a sequence of a first cellularnucleic acid molecule from the first cell or the first cell nucleus andthe first barcode sequence, and (ii) a second barcoded moleculecomprising a sequence of a second cellular nucleic acid molecule fromthe second cell or the second cell nucleus and the second barcodesequence.
 7. The method of claim 6, wherein each of the first cellularnucleic acid molecule and the second cellular nucleic acid molecule is adeoxyribonucleic acid (DNA) molecule.
 8. The method of claim 7, whereinthe DNA molecule is a genomic DNA (gDNA) molecule.
 9. The method ofclaim 6, wherein each of the first cellular nucleic acid molecule andthe second cellular nucleic acid molecule is a ribonucleic acid (RNA)molecule.
 10. The method of claim 9, wherein the RNA molecule is amessenger RNA (mRNA) molecule.
 11. The method of claim 1, wherein thefirst cell bead index nucleic acid molecule is embedded within the firsthydrogel, the first polymeric material, or the first cross-linkedmaterial, and wherein the second cell bead index nucleic acid moleculeis embedded within the second hydrogel, the second polymeric material,or the second cross-linked material.
 12. The method of claim 1, whereinthe first cell bead index nucleic acid molecule is covalently attachedto the first hydrogel, the first polymeric material, or the firstcross-linked material, and wherein the second cell bead index nucleicacid molecule is covalently attached to the second hydrogel, the secondpolymeric material, or the second cross-linked material.
 13. The methodof claim 12, wherein the first cell bead index nucleic acid molecule isreleasably attached to the first hydrogel, the first polymeric material,or the first cross-linked material, and wherein the second cell beadindex nucleic acid molecule is releasably attached to the secondhydrogel, the second polymeric material, or the second cross-linkedmaterial.
 14. The method of claim 13, wherein the first cell bead indexnucleic acid molecule is releasably attached to the first hydrogel, thefirst polymeric material, or the first cross-linked material by a firstlabile bond selected from the group consisting of a thermally labilebond, a chemically liable bond, an enzymatically labile bond, and aphotolabile bond, and wherein the second cell bead index nucleic acidmolecule is releasably attached to the second hydrogel, the secondpolymeric material, or the second cross-linked material by a secondlabile bond selected from the group consisting of a thermally labilebond, a chemically liable bond, an enzymatically labile bond, and aphotolabile bond.
 15. The method of claim 1, wherein each of the firstcell bead index nucleic acid molecule and the second cell bead indexnucleic acid molecule is selected from the group consisting of adouble-stranded nucleic acid molecule, a partially double-strandednucleic acid molecule, and a single-stranded nucleic acid molecule. 16.The method of claim 1, wherein the first cell bead index nucleic acidmolecule comprises a sequence complementary to a sequence of the firstnucleic acid barcode molecule, and wherein the second cell bead indexnucleic acid molecule comprises a sequence complementary to a sequenceof the second nucleic acid barcode molecule.
 17. The method of claim 16,wherein each of the first cell bead index nucleic acid molecule and thesecond cell bead index nucleic acid molecule comprises a poly-Asequence, and wherein each of the first nucleic acid barcode moleculeand the second nucleic acid barcode molecule comprises a poly-Tsequence.
 18. The method of claim 1, wherein the first nucleic acidbarcode molecule is attached to the first bead, and wherein the secondnucleic acid barcode molecule is attached to the second bead.
 19. Themethod of claim 18, wherein the first nucleic acid barcode molecule isreleasably attached to the first bead, and wherein the second nucleicacid barcode molecule is releasably attached to the second bead.
 20. Themethod of claim 18, wherein each of the first bead and the second beadis a gel bead.
 21. The method of claim 20, wherein the gel bead iscapable of being degraded.
 22. The method of claim 21, wherein the firstbead is capable of being degraded upon application of a first stimulusto the first bead, and wherein the second bead is capable of beingdegraded upon application of a second stimulus to the second bead. 23.The method of claim 1, wherein the first cell bead comprises the firstcell, and wherein the second cell bead comprises the second cell. 24.The method of claim 1, wherein the first cell bead comprises the firstcell nucleus, and wherein the second cell bead comprises the second cellnucleus.